Methods to induce targeted protein degradation through bifunctional molecules

ABSTRACT

The present application provides bifunctional compounds which act as protein degradation inducing moieties. The present application also relates to methods for the targeted degradation of endogenous proteins through the use of the bifunctional compounds that link a cereblon-binding moiety to a ligand that is capable of binding to the targeted protein which can be utilized in the treatment of proliferative disorders. The present application also provides methods for making compounds of the application and intermediates thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/264,266, filedJan. 31, 2019, which is a continuation of U.S. Ser. No. 15/816,646,filed Nov. 17, 2017, which is a continuation of U.S. Ser. No.15/148,253, filed May 6, 2016, now U.S. Pat. No. 9,821,068, issued Nov.21, 2017, which is a continuation of U.S. Ser. No. 14/707,930, filed May8, 2015, now U.S. Pat. No. 9,694,084, issued Jul. 4, 2017, which claimspriority to and the benefit of U.S. Ser. No. 62/096,318, filed on Dec.23, 2014, U.S. Ser. No. 62/128,457, filed on Mar. 4, 2015, and U.S. Ser.No. 62/149,170, filed on Apr. 17, 2015, the contents of each of whichare incorporated by reference in their entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Grant Nos. P01CA066996 and R01 CA176745 awarded by the National Institutes of Health.The government has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy of the Sequence Listing, created on Sep. 21,2020, is named “52095-701C09US—Sequence Listing_CON.txt” and is 3 KB insize.

BACKGROUND

Ubiquitin-Proteasome Pathway (UPP) is a critical pathway that regulateskey regulator proteins and degrades misfolded or abnormal proteins. UPPis central to multiple cellular processes, and if defective orimbalanced, it leads to pathogenesis of a variety of diseases. Thecovalent attachment of ubiquitin to specific protein substrates isachieved through the action of E3 ubiquitin ligases. These ligasescomprise over 500 different proteins and are categorized into multipleclasses defined by the structural element of their E3 functionalactivity.

Cereblon (CRBN) interacts with damaged DNA binding protein 1 and formsan E3 ubiquitin ligase complex with Cullin 4 where it functions as asubstrate receptor in which the proteins recognized by CRBN might beubiquitinated and degraded by proteasomes. Proteasome-mediateddegradation of unneeded or damaged proteins plays a very important rolein maintaining regular function of a cell, such as cell survival,proliferation and growth. A new role for CRBN has been identified, i.e.,the binding of immunomodulatory drugs (IMiDs), e.g. thalidomide, to CRBNhas now been associated with teratogenicity and also the cytotoxicity ofIMiDs, including lenalidomide, which are widely used to treat multiplemyeloma patients. CRBN is likely a key player in the binding,ubiquitination and degradation of factors involved in maintainingfunction of myeloma cells. These new findings regarding the role of CRBNin IMiD action stimulated intense investigation of CRBN's downstreamfactors involved in maintaining regular function of a cell (Chang X. IntJ Biochem Mol Biol. 2011; 2(3): 287-294).

The UPP is used to induce selective protein degradation, including useof fusion proteins to artificially ubiquitinate target proteins andsynthetic small-molecule probes to induce proteasome-dependentdegradation. Proteolysis Targeting Chimeras (PROTACs),heterobifunctional compounds composed of a target protein-binding ligandand an E3 ubiquitin ligase ligand, induced proteasome-mediateddegradation of selected proteins via their recruitment to E3 ubiquitinligase and subsequent ubiquitination. These drug-like molecules offerthe possibility of temporal control over protein expression. Suchcompounds are capable of inducing the inactivation of a protein ofinterest upon addition to cells or administration to an animal or human,and could be useful as biochemical reagents and lead to a new paradigmfor the treatment of diseases by removing pathogenic or oncogenicproteins (Crews C., Chemistry & Biology, 2010, 17(6):551-555;Schnnekloth J S Jr., Chembiochem, 2005, 6(1):40-46).

Successful treatment of various oncologic and immunologic disorders,such as cancer, is still a highly unmet need. Therefore, continueddevelopment of alternative approaches to cure or treat such disorders,including developing therapies involving protein degradation technology,remains of strong interest. Novel methods of more general nature thanexisting methods with respect to possible targets and different celllines or different in vivo systems could potentially lead to thedevelopment of future therapeutic treatments.

SUMMARY

The present application relates novel bifunctional compounds, whichfunction to recruit targeted proteins to E3 Ubiquitin Ligase fordegradation, and methods of preparation and uses thereof.

The present application further relates to targeted degradation ofproteins through the use of bifunctional molecules, includingbifunctional molecules that link a cereblon-binding moiety to a ligandthat binds the targeted protein.

The present application also relates to a bifunctional compound havingthe following structure:

-   -   Degron-Linker-Targeting Ligand,        wherein the Linker is covalently bound to at least one Degron        and at least one Targeting Ligand, the Degron is a compound        capable of binding to a ubiquitin ligase such as an E3 Ubiquitin        Ligase (e.g., cereblon), and the Targeting Ligand is capable of        binding to the targeted protein(s).

The present application also relates to a novel degradation inducingmoiety, or Degron, which is small in size and highly effective inrecruiting targeted proteins for degradation.

The present application further relates to a compound having Formula D,D1, or D3:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein

X, X₁, X₂, Y, R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m and n are each as definedherein.

The present application further relates to a Linker having Formula L0:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein p1, p2,p3, W, Q, and Z are each as defined herein, the Linker is covalentlybonded to the Degron with the

next to Q, and covalently bonded to the Targeting Ligand with the

next to Z.

The present application further relates to the Targeting Ligandsdescribed herein, including a compound of Formula TL-I to TL-VII.

The present application further relates to a bifunctional compound ofFormula X, I, or II

or an enantiomer, diastereomer, stereoisomer, or a pharmaceuticallyacceptable salt thereof, wherein the Linker is a group that covalentlybinds to the Targeting Ligand and Y, and the Targeting Ligand is capableof binding to or binds to a targeted protein, and wherein

X, X₁, X₂, Y, R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m and n are each as definedherein.

The present application further relates to a pharmaceutical formulationcomprising a therapeutically effective amount of a bifunctional compoundof Formula X, I, or II and a pharmaceutically acceptable carrier.

The present application also relates to a method for treating a diseaseor condition which is modulated by a targeted protein by administering atherapeutically effective amount of a bifunctional compound of FormulaX, I, or II to a subject in need thereof.

The present application also relates to use of a bifunctional compoundof Formula X, I, or II or a pharmaceutical formulation of theapplication for treating a disease or condition which is modulated by atargeted protein.

The present application also relates to use of a bifunctional compoundof Formula X, I, or II or a pharmaceutical formulation of theapplication in the manufacture of a medicament for treating a disease orcondition which is modulated by a targeted protein.

The present application also relates to methods for treating cancer byadministering a therapeutically effective amount of a bifunctionalcompound of Formula X, I, or II to a subject in need thereof.

The present application also relates to use of a bifunctional compoundof Formula X, I, or II or a pharmaceutical formulation of theapplication for treating cancer.

The present application also relates to use of a bifunctional compoundof Formula X, I, or II or a pharmaceutical formulation of theapplication in the manufacture of a medicament for treating cancer.

The compounds and methods of the present application address unmet needsin the treatment of diseases or disorders in which pathogenic oroncogenic endogenous proteins play a role, such as, for example, cancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this application belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent application, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed application. Inthe case of conflict, the present specification, including definitions,will control. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the present application will beapparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe application, will be better understood when read in conjunction withthe appended drawings.

FIGS. 1A-1H: Design and characterization of dBET1. FIG. 1A shows thechemical structure of JQ1(S), JQ1(R) and the phthalimides; FIG. 1B showsthe chemical structure of dBET1; FIG. 1C shows DMSO normalized BRD4binding signal measured by AlphaScreen for the indicated compounds(values represent mean±stdev of triplicate analysis); FIG. 1D shows thecrystal structure of dBET1 bound to bromodomain 1 of BRD4; FIG. 1E showsdocking of the structure in FIG. 1D into the published DDB1-CRBNstructure; FIG. 1F shows immunoblot analysis for BRD4 and Vinculin after18 h treatment of MV4-11 cells with the indicated concentrations ofdBET1; FIG. 1G shows the crystal structure of dBET1 bound to BRD4overlaid with the structure of JQ1 bound to BRD4; FIG. 1H is a Westernblot illustrating the degree of degradation of BRD4 and reduction inc-MYC by treatment of cells with increased concentrations of abifunctional compound of the application, dBET1.

FIGS. 2A-2Q: FIG. 2A shows the immunoblot analysis for BRD4 aftertreatment of MV4-11 cells with the indicated concentrations of dBET1(R)for 18 h; FIG. 2B shows cell count normalized BRD4 levels as determinedby high-content assay in SUM149 cells treated with the indicatedconcentrations of dBET1 and dBET1(R) for 18 h (values representmean±stdev of triplicate analysis, normalized to DMSO treated cells andbaseline corrected based on immunoblots in FIG. 2C); FIG. 2C showsimmunoblot analysis for BRD4 and Vinculin after treatment of SUM149cells with the indicated concentrations of dBET1 and dBET1(R) for 18 h;FIG. 2D shows immunoblot analysis for BRD4 and Vinculin after treatmentof MV4-11 cells with 100 nM dBET1 at the indicated time points; FIG. 2Eshows cellular viability dose-response of dBET1 and JQ1 treatment for 24h in MV4-11 as determined ATP levels (values represent mean±stdev ofquadruplicate analysis); FIG. 2F is a bar graph depiction of foldincrease of apoptosis assessed via caspase glo assay relative to DMSOtreated controls after 24 h treatment in MV4-11 or DHL4 cells (valuesrepresent mean±stdev of quadruplicate analysis); FIG. 2G showsimmunoblot analysis for BRD4 and Vinculin after treatment of primarypatient cells with the indicated concentrations of dBET1 for 24 hours;FIG. 2H is a bar graph depiction of fraction of Annexin V positiveprimary patient cells after 24 h treatment with either dBET1 or JQ1 atthe indicated concentrations (values represent the average of duplicatesand the range as error bars) (representative counter plots shown inFIGS. 2L and 2M); FIGS. 2I and 2J are immunoblot analysis showing BRD4and Vinculin 18 h after dBET1 treatment with the indicatedconcentrations in SUM159 and MOLM13, respectively; FIG. 2K showsimmunoblot analysis for BRD2, BRD3, and BRD4 and c-Myc at different timepoints after treatment of cells with the 100 nM of dBET1; FIG. 2L are aseries of flow cytometry plots illustrating Annexin V/PI data of primarypatient cells treated with the indicated concentrations of JQ1 and dBET1for 24 h; FIG. 2M is a bar graph depiction of Annexin V positive MV4-11cells after treatment with dBET1 or JQ1 at the indicated concentrations(data represents mean±stdev of triplicate analysis); FIG. 2N is animmunoblot analysis of cleaved caspase 3, PARP cleavage and vinculinafter treatment with dBET1 and JQ1 at the indicated concentrations for24 h; FIG. 2O are bar graphs illustrating the kinetic advantage of BETdegradation on apoptosis (Caspase-GLO) relative to treated controls:MV4-1 cells were treated for 4 or 8 h with JQ1 or dBET1 at the indicatedconcentrations, followed by drug wash-out before being plated indrug-free media for 24 h; FIG. 2P is a bar graph depiction of foldincrease of apoptosis of MV4-11 cells treated for 4 or 8 h with JQ1 ordBET1 at the indicated concentrations, relative to DMSO treated controlsassessed via caspase glo assay (drug were washed out with PBS (3×)before being plated in drug-free media for a final treatment duration of24 h); FIG. 2Q is an immunoblot analysis of cleaved caspase 3, PARPcleavage and vinculin after identical treatment conditions in FIG. 2P.

FIGS. 3A-3F: FIGS. 3A-3D show chemical and genetic rescue ofdBET1-mediated degradation of BRD4. FIG. 3A shows immunoblot analysisfor BRD4 and Vinculin after treatment of MV4-11 cells with the indicatedconcentrations of dBET1, JQ1, and thalidomide for 24 h; FIG. 3B showsimmunoblot analysis for BRD4 and Vinculin after 4 h pre-treatment witheither DMSO, Carfilzomib (0.4 μM), JQ1 (10 μM) or thalidomide (10 μM)followed by 2 h dBET1 treatment at a concentration of 100 nM; FIG. 3Cshows immunoblot analysis for BRD4 and Vinculin after a 4 hpre-treatment with 1 μM MLN4924 followed by 2 h dBET1 treatment at theindicated concentrations; FIG. 3D shows immunoblot analysis for BRD4,CRBN and tubulin after treatment of MM1S^(WT) or MM1S^(CRBN−/−) withdBET1 for 18 h at the indicated concentrations;

FIG. 3E is an immunoblot analysis comparing the concentration of BRD4 incells treated with various concentrations of thalidomide, JQ1, anddBET1; FIG. 3F is an immunoblot analysis showing the concentration ofBRD4 in cells treated with carfilzomib (400 nM), JQ1 (20 uM), orthalidomide (20 uM) for 4 hours and with dBET1 (100 nM) as indicated for2 hours.

FIGS. 4A-4F: FIGS. 4A-4E show selective BET bromodomain degradation byexpression proteomics: MV4-11 cells were treated for 2 hours with DMSO,250 nM dBET1 or 250 nM JQ1. FIG. 4A depicts fold change of abundance of7429 proteins comparing JQ1 to DMSO treatment as well as theirrespective p-value (T-test) (data from triplicate analysis); FIG. 4Bdepicts fold change of abundance of 7429 proteins comparing 250 nM dBET1to DMSO treatment (data from triplicate analysis); FIG. 4C is a bargraph depiction of changes in protein levels of the selected proteins asshown normalized to DMSO (values represent mean±stdev of triplicates);FIG. 4D shows immunoblot analysis of BRD2, BRD3, BRD4, MYC, PIM1 andVINC after 2 h treatment of MV4-11 cells with either DMSO, 250 nM dBET1or 250 nM JQ1; FIG. 4E is a bar graph depiction of qRT-PCR analysis oftranscript levels of BRD2, BRD3, BRD4, MYC and PIM1 after 2 h treatmentof MV4-11 cells with either DMSO, 250 nM dBET1 or 250 nM JQ1 (valuesrepresent mean+/−stdev of triplicates); FIG. 4F is an immunoblotanalysis for IKZF3 and Vinculin after 24 h treatment with thalidomide orthe indicated concentrations of dBET1 in MM1S cell line.

FIGS. 5A-5B: FIG. 5A is a Western blot showing the concentration of BRD4in cells treated with various concentrations of JQ1-Rev, in comparisonwith 100 nM of a bifunctional compound of the application, dBET1. FIG.5B is a drawing of the chemical structure of JQ1-Rev (JQI-11-079).

FIGS. 6A-6B: FIGS. 6A and 6B are a series of graphs that illustrate thetranscription levels of BRD4 assayed via qRT-PCR after 2 hrs (FIG. 6A)or 4 hrs (FIG. 6B) from cells treated with various concentrations of JQ1or dBET.

FIG. 7 is a Western blot illustrating the degree of degradation of BRD4by treatment of a human cell line MM1S and a human cell line MM1S thatis deficient in cereblon with increased concentrations of a bifunctionalcompound of the application, dBET1.

FIG. 8 is a Western blot illustrating the degree of degradation of BRD4by treatment of cells with increased concentrations of a bifunctionalcompound of the application, dBET2.

FIGS. 9A-9B: FIG. 9A is a Western blot illustrating reduction in PLK1 bytreatment of cells with increased concentrations of a bifunctionalcompound of the application, dBET2; FIG. 9B is a bar graph depicting PLKintensity in dBET2 treated cells as percentage of that in DMSO treatedcells.

FIGS. 10A-10D: dFKBP-1 and dFKBP-2 mediated degradation of FKBP12. FIG.10A depicts chemical structures of dFKBP-1 and dFKBP-2; FIG. 10Billustrates immunoblot analysis for FKBP12 and Vinculin after 18 htreatment with the indicated compounds; FIG. 10C shows immunoblotanalysis for FKBP12 and Vinculin after a 4 h pre-treatment with eitherDMSO, Carfilzomib (400 nM), MLN4924 (1 μM), SLF (20 μM) or thalidomide(10 μM) followed by a 4 h dFKBP-1 treatment at a concentration of 1 μMin MV4-11 cells; FIG. 10D shows immunoblot analysis for FKBP12, CRBN andtubulin after treatment of 293FT^(WT) or 293FT^(CRBN−/−) with dFKBP12 atthe indicated concentrations for 18 h.

FIGS. 11A-11C: FIG. 11A is a diagram showing selectivity of dBET1 forbinding to BETs over other human bromodomains, as determined by singlepoint screening (BromoScan); FIG. 11B shows results from a dimerizationassay measuring dBET1 induced proximity (at 111 nM) between recombinantBRD4 bromodomain and recombinant CRBN-DDB1 (values represent mean±stdevof quadruplicate analysis and are normalized to DMSO); FIG. 11C is a bargraph showing the competition of dBET1 induced proximity in the presenceof DMSO (vehicle), JQ1(S), thal-(−), JQ1(R) and thal-(+), all at a finalconcentration of 1 μM (values represent mean ±stdev of quadruplicateanalysis and are normalized to DMSO).

FIGS. 12A-12E: FIG. 12A is a bar graph showing the tumor volume ofvehicle treated mice (n=5) or mice treated with dBET1 at a concentrationof 50 mg/kg (n=6) over a treatment period of 14 days; FIG. 12B is a bargraph comparing the tumor weight after termination of the experimentshown in FIG. 12A on day 14; FIG. 12C is an immunoblot analysis forBRD4, MYC and Vinculin using tumor lysates from mice treated either oncefor 4 h or twice for 22 h and 4 h compared to a vehicle treated control;FIG. 12D shows immunohistochemistry staining for BRD4, MYC and Ki67 of arepresentative tumor of a dBET1 treated and a control treated mouse;FIG. 12E is a bar graph depicting quantification of the staining in FIG.12D based on 3 independent areas within that section (data representsmean±stdev of triplicate analysis and is normalized to DMSO).

FIGS. 13A-13D: Pharmacokinetic studies of dBET1 in CD1 mice. FIG. 13A isa graph showing concentration of dBET1 in CD1 mice followingintraperitoneal injection of dBET1 formulated in 0.5% MC in water; FIG.13B is a table depicting pharmacokinetic parameters in mice from FIG.13A; FIG. 13C is a graph showing the change in weight of mice treatedwith 50 mg/kg q.d. of dBET1 or vehicle; FIG. 13D are bar graphs showingchanges in HTC, WBC, or PLT in mice from FIG. 13C.

FIGS. 14A-14BB: High content assay measuring BRD4 levels in cells(293FT^(WT) or 293FT^(CRBN−/−)) after 4 hour treatment with indicatedconcentrations of the bifunctional compounds of the present application.FIGS. 14A-14B: BRD4 levels in 293FT^(WT) (FIG. 14A) or 293FT^(CRBN−/−)(FIG. 14B) after 4 hour treatment with indicated concentrations of JQ1;FIGS. 14C-14D: BRD4 levels in 293FT^(WT) (FIG. 14C) or 293FT^(CRBN−/−)(FIG. 14D) after 4 hour treatment with indicated concentrations ofdBET1; FIG. 14E-14F: BRD4 levels in 293FT^(WT) (FIG. 14E) or293FT^(CRBN−/−) (FIG. 14F) after 4 hour treatment with indicatedconcentrations of dBET2; FIGS. 14G-14H: BRD4 levels in 293FT^(WT) (FIG.14G) or 293FT^(CRBN−/−) (FIG. 14H) after 4 hour treatment with indicatedconcentrations of dBET3; FIGS. 14I-14J: BRD4 levels in 293FT^(WT) (FIG.14 ) or 293FT^(CRBN)-(FIG. 14J) after 4 hour treatment with indicatedconcentrations of dBET4; FIGS. 14K-14L: BRD4 levels in 293FT^(WT) (FIG.14K) or 293FT^(CRBN−/−) (FIG. 14L) after 4 hour treatment with indicatedconcentrations of dBET5; FIGS. 14M-14N: BRD4 levels in 293FT^(WT) (FIG.14M) or 293FT^(CRBN−/−) (FIG. 14N) after 4 hour treatment with indicatedconcentrations of dBET6; FIGS. 140-14P: BRD4 levels in 293FT^(WT) (FIG.14O) or 293FT^(CRBN−/−) (FIG. 14P) after 4 hour treatment with indicatedconcentrations of dBET7; FIGS. 14Q-14R: BRD4 levels in 293FT^(WT) (FIG.14Q) or 293FT^(CRBN−/−) (FIG. 14R) after 4 hour treatment with indicatedconcentrations of dBET8; FIGS. 14S-14T: BRD4 levels in 293FT^(WT) (FIG.14S) or 293FT^(CRBN−/−) (FIG. 14T) after 4 hour treatment with indicatedconcentrations of dBET9; FIGS. 14U-14V: BRD4 levels in 293FT^(WT) (FIG.14U) or 293FT^(CRBN−/−) (FIG. 14V) after 4 hour treatment with indicatedconcentrations of dBET10; FIGS. 14W-14X: BRD4 levels in 293FT^(WT) (FIG.14W) or 293FT^(CRBN−/−) (FIG. 14X) after 4 hour treatment with indicatedconcentrations of dBET15; FIGS. 14Y-14Z: BRD4 levels in 293FT^(WT) (FIG.14Y) or 293FT^(CRBN−/−) (FIG. 14Z) after 4 hour treatment with indicatedconcentrations of dBET17; FIGS. 14AA-14BB: BRD4 levels in 293FT^(WT)(FIG. 14AA) or 293FT^(CRBN−/−) (FIG. 14BB) after 4 hour treatment withindicated concentrations of dBET18.

FIGS. 15A-15F: Immunoblots of BRD levels in cells treated with varyingconcentrations of the bifunctional compounds of the present application.FIG. 15A is an immunoblot showing BRD4 levels in BAF3_K-RAS cellstreated with the indicated concentrations of dBET1 or dBET6 for 16.5hours; FIG. 15B is an immunoblot showing BRD4 levels in BAF3_K-RAS orSEMK2 cells treated with the indicated concentrations of dBET1 for 16.5hours; FIG. 15C is an immunoblot showing BRD4 levels in SEMK2 cellstreated with the indicated concentrations of dBET1 or dBET6 for 16.5hours; FIG. 15D is an immunoblot showing BRD4 levels in Monomac1 cellstreated with the indicated concentrations of dBET1 or dBET6 for 16hours; FIG. 15E is an immunoblot showing levels of BRD4, BRD2, and BRD3at various time points in MV4-11 cells treated with 50 nM of dBET6; FIG.15F is an immunoblot showing BRD4 levels in MM1S^(W)T or MM1S^(CRBN−/−)cells treated with the indicated concentrations of dBET6 for 16 hours.

FIGS. 16A-16B: Immunoblots of protein levels in cells treated with thebifunctional compounds of the present application. FIG. 16A is animmunoblot showing levels of BRD4 and PLK1 in cells (WT or CRBN−/−)treated with 1 μM of dBET2, dBET7, dBET8, or dBET10; FIG. 16B is animmunoblot showing BRD4 levels at the indicated time points after thecells were treated with 100 nM dBET18.

FIGS. 17A-17E: Cell viability after treatment with the bifunctionalcompounds of the present application. FIGS. 17A-17B indicate cellviability EC₅₀ values of the bifunctional compounds of the presentapplication in various cells lines; FIGS. 17C-17E show cell viabilityafter treatment with increasing concentrations of JQ1, dBET1, dBET6,dBET7, or dBET8.

FIGS. 18A-18C shows viability of MOLT4 (FIG. 18A), DND41 (FIG. 18B), andCUTLL1 (FIG. 18C) cells after being treated with increasingconcentrations of dBET compounds.

FIG. 19 is an immunoblot showing GR levels in cells treated withindicated concentrations of dGR3.

DETAILED DESCRIPTION

Small-molecule antagonists disable discrete biochemical properties ofthe protein targets. For multi-domain protein targets, the pharmacologicconsequence of drug action is limited by selective disruption of onedomain-specific activity. Also, target inhibition is kinetically limitedby the durability and degree of the target engagement. These features oftraditional drug molecules are challenging to the development ofinhibitors targeting transcription factors and chromatin-associatedepigenetic proteins, which function as multi-domain biomolecularscaffolds and generally feature rapid association and dissociationkinetics. A chemical strategy was devised to prompt ligand-dependenttarget protein degradation via chemical conjugation with derivatizedphthalimides that hijack the function of the Cereblon E3 ubiquitinligase complex. Using this approach, an acetyl-lysine competitiveantagonist that displaces BET bromodomains from chromatin (JQ1) wasconverted to a phthalimide-conjugated ligand that prompts immediateCereblon-dependent BET protein degradation (dBET1). Expressionproteomics confirmed high specificity for BET family members BRD2, BRD3and BRD4 among 7429 proteins detected. Degradation of BET bromodomainsis associated with a more rapid and robust apoptotic response comparedto bromodomain inhibition in primary human leukemic blasts and in humanleukemia xenograft in vivo. Following this approach, additional seriesof phthalimide-conjugated ligands targeting other proteins fordegradation, such as FKBP12, were also developed. A facile new strategyto control the stability of target proteins, including previouslyintractable protein targets, is described herein.

As discussed above, there remains a need for the development of noveltherapies for the treatment of various disorders, including oncologicand immunologic disorders. The present application provides novelcompounds of general Formula I, which induce formation of a complexbetween two proteins to result in a desired biological effect. Thesecompounds can be prepared by using traditional synthetic methods andused by means of addition to a subject as drugs.

The present application provides a novel class of compounds useful forthe treatment of cancer and other proliferative conditions.

The present application relates to small molecule E3 ligase ligands(Degrons) which are covalently linked to a targeted protein ligandthrough a Linker of varying length and functionality, which can be usedas therapeutics for treating various diseases including cancer. Thepresent application also relates to a technology platform of bringingtargeted proteins to E3 ligases, for example CRBN, for ubiquitinationand subsequent proteasomal degradation using the bifunctional smallmolecules comprising a thalidomide-like Degron and a Targeting Ligandconnected to each other via a Linker.

This technology platform provides therapies based upon depression oftarget protein levels by degradation. The novel technology allows fortargeted degradation to occur in a more general nature than existingmethods with respect to possible targets and different cell lines ordifferent in vivo systems.

Compounds of the present application may offer important clinicalbenefits to patients, in particular for the treatment of the diseasestates and conditions modulated by the proteins of interest.

Compounds of the Application

The present application relates to bifunctional compounds which findutility as modulators of ubiquitination and proteosomal degradation oftargeted proteins, especially compounds comprising an inhibitor of apolypeptide or a protein that is degraded and/or otherwise inhibited bythe bifunctional compounds of the present application. In particular,the present application is directed to compounds which contain a ligand,e.g., a small molecule ligand (i.e., having a molecular weight of below2,000, 1,000, 500, or 200 Daltons), such as a thalidomide-like ligand,which is capable of binding to a ubiquitin ligase, such as cereblon, anda moiety that is capable of binding to a target protein, in such a waythat the target protein is placed in proximity to the ubiquitin ligaseto effect degradation (and/or inhibition) of that protein.

In general, the present application provides compounds having thegeneral structure:

-   -   Degron-Linker-Targeting Ligand,        wherein the Linker is covalently bound to at least one Degron        and at least one Targeting Ligand, the Degron is a compound        capable of binding to a ubiquitin ligase such as an E3 Ubiquitin        Ligase (e.g., cereblon), and the Targeting Ligand is capable of        binding to the targeted protein(s).

In certain embodiments, the present application provides a compound ofFormula X:

or an enantiomer, diastereomer, stereoisomer, or a pharmaceuticallyacceptable salt thereof, wherein:

-   -   the Linker is a group that covalently binds to the Targeting        Ligand and Y; and    -   the Targeting Ligand is capable of binding to or binds to a        targeted protein;        and wherein

X, X₁, X₂, Y, R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m and n are each as definedherein.

In certain embodiments, the present application provides a compound ofFormula I:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof, wherein:

-   -   the Linker is a group that covalently binds to the Targeting        Ligand and Y; and    -   the Targeting Ligand is capable of binding to or binds to a        targeted protein;        and wherein X, XY, R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m and n are        each as defined herein.

In certain embodiments, the present application provides a compound ofFormula I:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof, wherein:

-   -   the Linker is a group that covalently binds to the Targeting        Ligand and Y; and    -   the Targeting Ligand is capable of binding to or binds to a        targeted protein;        and wherein X₁, X₂, Y, R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m and n are        each as defined herein.        Degron

The Degron is a compound that serves to link a targeted protein, throughthe Linker and Targeting Ligand, to a ubiquitin ligase for proteosomaldegradation. In certain embodiments, the Degron is a compound that iscapable of binding to or binds to a ubiquitin ligase. In furtherembodiments, the Degron is a compound that is capable of binding to orbinds to a E3 Ubiquitin Ligase. In further embodiments, the Degron is acompound that is capable of binding to or binds to cereblon. In furtherembodiments, the Degron is a thalidomide or a derivative or analogthereof.

In certain embodiments, the Degron is a compound having Formula D:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

is

Y is a bond, (CH₂)₁₋₆, (CH₂)₀₋₆—O, (CH₂)₀₋₆—C(O)NR₂′, (CH₂)₀₋₆—NR₂′C(O),(CH₂)₀₋₆—NH, or (CH₂)₀₋₆—NR₂;

X is C(O) or C(R₃)₂;

X₁-X₂ is C(R₃)═N or C(R₃)₂—C(R₃)₂;

each R₁ is independently halogen, OH, C₁-C₆ alkyl, or C₁-C₆ alkoxy;

R₂ is C₁-C₆ alkyl, C(O)—C₁-C₆ alkyl, or C(O)—C₃-C₆ cycloalkyl;

R₂′ is H or C₁-C₆ alkyl;

each R₃ is independently H or C₁-C₃ alkyl;

each R₃′ is independently C₁-C₃ alkyl;

each R₄ is independently H or C₁-C₃ alkyl; or two R₄, together with thecarbon atom to which they are attached, form C(O), a C₃-C₆ carbocycle,or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatomsselected from N and O;

R₅ is H, deuterium, C₁-C₃ alkyl, F, or C₁;

m is 0, 1, 2 or 3; and

n is 0, 1 or 2;

wherein the compound is covalently bonded to another moiety (e.g., acompound, or a Linker) via

In certain embodiments, the Degron is a compound of Formula D, wherein

is

In certain embodiments, the Degron is a compound of Formula D, wherein

is

In certain embodiments, the Degron is a compound of Formula D, wherein Xis C(O).

In certain embodiments, the Degron is a compound of Formula D, wherein Xis C(R₃)₂; and each R₃ is H. In certain embodiments, X is C(R₃)₂; andone of R₃ is H, and the other is C₁-C₃ alkyl selected from methyl,ethyl, and propyl. In certain embodiments, X is C(R₃)₂; and each R₃ isindependently selected from methyl, ethyl, and propyl.

In certain embodiments, the Degron is a compound of Formula D, whereinX₁-X₂ is C(R₃)═N. In certain embodiments, X₁-X₂ is CH═N. In certainembodiments, X₁-X₂ is C(R₃)═N; and R₃ is C₁-C₃ alkyl selected frommethyl, ethyl, and propyl. In certain embodiments, X₁-X₂ is C(CH₃)═N.

In certain embodiments, the Degron is a compound of Formula D, whereinX₁-X₂ is C(R₃)₂—C(R₃)₂; and each R₃ is H. In certain embodiments, X₁-X₂is C(R₃)₂—C(R₃)₂; and one of R₃ is H, and the other three R₃ areindependently C₁-C₃ alkyl selected from methyl, ethyl, and propyl. Incertain embodiments, X₁-X₂ is C(R₃)₂—C(R₃)₂; and two of the R₃ are H,and the other two R₃ are independently C₁-C₃ alkyl selected from methyl,ethyl, and propyl. In certain embodiments, X₁-X₂ is C(R₃)₂—C(R₃)₂; andthree of the R₃ are H, and the remaining R₃ is C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In certain embodiments, the Degron is a compound of Formula D, wherein Yis a bond.

In certain embodiments, the Degron is a compound of Formula D, wherein Yis (CH₂)₁, (CH₂)₂, (CH₂)₃, (CH₂)₄, (CH₂), or (CH₂)₆. In certainembodiments, Y is (CH₂)₁, (CH₂)₂, or (CH₂)₃. In certain embodiments, Yis (CH₂)₁ or (CH₂)₂.

In certain embodiments, the Degron is a compound of Formula D, wherein Yis O, CH₂—O, (CH₂)₂—O, (CH₂)₃—O, (CH₂)₄—O, (CH₂)₅—O, or (CH₂)₆—O. Incertain embodiments, Y is O, CH₂—O, (CH₂)₂—O, or (CH₂)₃—O. In certainembodiments, Y is O or CH₂—O. In certain embodiments, Y is O.

In certain embodiments, the Degron is a compound of Formula D, wherein Yis C(O)NR₂′, CH₂—C(O)NR₂′, (CH₂)₂—C(O)NR₂′, (CH₂)₃—C(O)NR₂′,(CH₂)₄—C(O)NR₂′, (CH₂)₅—C(O)NR₂′, or (CH₂)₆—C(O)NR₂′. In certainembodiments, Y is C(O)NR₂′, CH₂—C(O)NR₂′, (CH₂)₂—C(O)NR₂′, or(CH₂)₃—C(O)NR₂′. In certain embodiments, Y is C(O)NR₂′ or CH₂—C(O)NR₂′.In certain embodiments, Y is C(O)NR_(2′.)

In certain embodiments, the Degron is a compound of Formula D, wherein Yis NR₂′C(O), CH₂—NR₂′C(O), (CH₂)₂—NR₂′C(O), (CH₂)₃—NR₂′C(O),(CH₂)₄—NR₂′C(O), (CH₂)₅—NR₂′C(O), or (CH₂)₆—NR₂′C(O). In certainembodiments, Y is NR₂′C(O), CH₂—NR₂′C(O), (CH₂)₂—NR₂′C(O), or(CH₂)₃—NR₂′C(O). In certain embodiments, Y is NR₂′C(O) or CH₂—NR₂′C(O).In certain embodiments, Y is NR₂′C(O).

In certain embodiments, the Degron is a compound of Formula D, whereinR₂′ is H. In certain embodiments, the Degron is a compound of Formula D,wherein R₂′ is selected from methyl, ethyl, propyl, butyl, i-butyl,t-butyl, pentyl, i-pentyl, and hexyl. In certain embodiments, R₂′ isC₁-C₃ alkyl selected from methyl, ethyl, and propyl.

In certain embodiments, the Degron is a compound of Formula D, wherein Yis NH, CH₂—NH, (CH₂)₂—NH, (CH₂)₃—NH, (CH₂)₄—NH, (CH₂)—NH, or (CH₂)₆—NH.In certain embodiments, Y is NH, CH₂—NH, (CH₂)₂—NH, or (CH₂)₃—NH. Incertain embodiments, Y is NH or CH₂—NH. In certain embodiments, Y is NH.

In certain embodiments, the Degron is a compound of Formula D, wherein Yis NR₂, CH₂—NR₂, (CH₂)₂—NR₂, (CH₂)₃—NR₂, (CH₂)₄—NR₂, (CH₂)—NR₂, or(CH₂)₆—NR₂. In certain embodiments, Y is NR₂, CH₂—NR₂, (CH₂)₂—NR₂, or(CH₂)₃—NR₂. In certain embodiments, Y is NR₂ or CH₂—NR₂. In certainembodiments, Y is NR₂.

In certain embodiments, the Degron is a compound of Formula D, whereinR₂ is selected from methyl, ethyl, propyl, butyl, i-butyl, t-butyl,pentyl, i-pentyl, and hexyl. In certain embodiments, R₂ is C₁-C₃ alkylselected from methyl, ethyl, and propyl.

In certain embodiments, the Degron is a compound of Formula D, whereinR₂ is selected from C(O)-methyl, C(O)-ethyl, C(O)-propyl, C(O)-butyl,C(O)-i-butyl, C(O)-t-butyl, C(O)— pentyl, C(O)-i-pentyl, and C(O)-hexyl.In certain embodiments, R₂ is C(O)—C₁-C₃ alkyl selected fromC(O)-methyl, C(O)-ethyl, and C(O)-propyl.

In certain embodiments, the Degron is a compound of Formula D, whereinR₂ is selected from C(O)-cyclopropyl, C(O)-cyclobutyl, C(O)-cyclopentyl,and C(O)-cyclohexyl. In certain embodiments, R₂ is C(O)-cyclopropyl.

In certain embodiments, the Degron is a compound of Formula D, whereinR₃ is H.

In certain embodiments, the Degron is a compound of Formula D, whereinR₃ is C₁-C₃ alkyl selected from methyl, ethyl, and propyl. In certainembodiments, R₃ is methyl.

In certain embodiments, the Degron is a compound of Formula D, wherein nis 0.

In certain embodiments, the Degron is a compound of Formula D, wherein nis 1.

In certain embodiments, the Degron is a compound of Formula D, wherein nis 2.

In certain embodiments, the Degron is a compound of Formula D, whereineach R₃′ is independently C₁-C₃ alkyl selected from methyl, ethyl, andpropyl.

In certain embodiments, the Degron is a compound of Formula D, wherein mis 0.

In certain embodiments, the Degron is a compound of Formula D, wherein mis 1.

In certain embodiments, the Degron is a compound of Formula D, wherein mis 2.

In certain embodiments, the Degron is a compound of Formula D, wherein mis 3.

In certain embodiments, the Degron is a compound of Formula D, whereineach R₁ is independently selected from halogen (e.g., F, Cl, Br, and I),OH, C₁-C₆ alkyl (e.g., methyl, ethyl, propyl, butyl, i-butyl, t-butyl,pentyl, i-pentyl, and hexyl), and C₁-C₆ alkoxy (e.g., methoxy, ethoxy,propoxy, butoxy, i-butoxy, t-butoxy, and pentoxy). In furtherembodiments, the Degron is a compound of Formula D, wherein each R₁ isindependently selected from F, Cl, OH, methyl, ethyl, propyl, butyl,i-butyl, t-butyl, methoxy, and ethoxy.

In certain embodiments, the Degron is a compound of Formula D, whereineach R₄ is H.

In certain embodiments, the Degron is a compound of Formula D, whereinone of R₄ is H, and the other R₄ is C₁-C₃ alkyl selected from methyl,ethyl, and propyl.

In certain embodiments, the Degron is a compound of Formula D, whereineach R₄ is independently C₁-C₃ alkyl selected from methyl, ethyl, andpropyl.

In certain embodiments, the Degron is a compound of Formula D, whereintwo R₄, together with the carbon atom to which they are attached, formC(O).

In certain embodiments, the Degron is a compound of Formula D, whereintwo R₄, together with the carbon atom to which they are attached, formcyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

In certain embodiments, the Degron is a compound of Formula D, whereintwo R₄, together with the carbon atom to which they are attached, form a4-, 5-, or 6-membered heterocycle selected from oxetane, azetidine,tetrahydrofuran, pyrrolidine, piperidine, piperazine, and morpholine. Incertain embodiments, two R₄, together with the carbon atom to which theyare attached, form oxetane.

In certain embodiments, the Degron is a compound of Formula D, whereinR₅ is H, deuterium, or C₁-C₃ alkyl. In further embodiments, R₅ is in the(S) or (R) configuration. In further embodiments, R₅ is in the (S)configuration. In certain embodiments, the Degron is a compound ofFormula D, wherein the compound comprises a racemic mixture of (S)—R₅and (R)—R₅.

In certain embodiments, the Degron is a compound of Formula D, whereinR₅ is H.

In certain embodiments, the Degron is a compound of Formula D, whereinR₅ is deuterium.

In certain embodiments, the Degron is a compound of Formula D, whereinR₅ is C₁-C₃ alkyl selected from methyl, ethyl, and propyl. In certainembodiments, R₅ is methyl.

In certain embodiments, the Degron is a compound of Formula D, whereinR₅ is F or Cl.

In further embodiments, R₅ is in the (S) or (R) configuration. Infurther embodiments, R₅ is in the (R) configuration. In certainembodiments, the Degron is a compound of Formula D, wherein the compoundcomprises a racemic mixture of (S)—R₅ and (R)—R₅. In certainembodiments, R₅ is F.

Each of the moieties defined for one of

X, X₁, X₂, Y, R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m, and n, can be combinedwith any of the moieties defined for the others of

X, X₁, X₂, Y, R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m, and n.

In certain embodiments, the Degron is a compound having Formula D1:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein X, Y,R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m, and n are each as defined above inFormula D.

Each of X, Y, R, R₂, R₂′, R₃, R₃′, R₄, R₅, m, and n can be selected fromthe moieties described above in Formula D. Each of the moieties definedfor one of X, Y, R, R₂, R₂′, R₃, R₃′, R₄, R₅, m, and n, can be combinedwith any of the moieties defined for the others of X, Y, R₁, R₂, R₂′,R₃, R₃′, R₄, R₅, m, and n, as described above in Formula D.

In certain embodiments, the Degron is a compound of Formula D2:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein each ofR, R₃′, m and n is as defined above and can be selected from anymoieties or combinations thereof described above.

In certain embodiments, the Degron is a compound of the followingstructure:

or an enantiomer, diastereomer, or stereoisomer thereof.

In certain embodiments, the Degron is a compound of Formula D3:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein X₁, X₂,Y, R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m, and n are each as defined above inFormula D.

Each of X₁, X₂, Y, R, R₂, R₂′, R₃, R₃′, R₄, R₅, m, and n can be selectedfrom the moieties described above in Formula D. Each of the moietiesdefined for one of X₁, X₂, Y, R, R₂, R₂′, R₃, R₃′, R₄, R₅, m, and n, canbe combined with any of the moieties defined for the others of X₁, X₂,Y, R₁, R₂, R₂′, R₃, R₃′, R₄, R₅, m, and n, as described above in FormulaD.

In certain embodiments, the Degron is selected from the following,wherein X is H, deuterium, C₁-C₃ alkyl, or halogen; and R is a Linker:

In certain embodiments, the Degron is selected from the following:

In certain embodiments, the Degron is selected from the following:

Linker

The Linker is a bond or a carbon chain that serves to link a TargetingLigand with a Degron. In certain embodiments, the carbon chainoptionally comprises one, two, three, or more heteroatoms selected fromN, O, and S. In certain embodiments, the carbon chain comprises onlysaturated chain carbon atoms. In certain embodiments, the carbon chainoptionally comprises two or more unsaturated chain carbon atoms (e.g.,C═C or C≡C). In certain embodiments, one or more chain carbon atoms inthe carbon chain are optionally substituted with one or moresubstituents (e.g., oxo, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₃ alkoxy, OH, halogen, NH₂, NH(C₁-C₃ alkyl), N(C₁-C₃ alkyl)₂, CN,C₃-C₈ cycloalkyl, heterocyclyl, phenyl, and heteroaryl).

In certain embodiments, the Linker comprises at least 5 chain atoms(e.g., C, O, N, and S). In certain embodiments, the Linker comprisesless than 20 chain atoms (e.g., C, O, N, and S). In certain embodiments,the Linker comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,or 19 chain atoms (e.g., C, O, N, and S). In certain embodiments, theLinker comprises 5, 7, 9, 11, 13, 15, 17, or 19 chain atoms (e.g., C, O,N, and S). In certain embodiments, the Linker comprises 5, 7, 9, or 11chain atoms (e.g., C, O, N, and S). In certain embodiments, the Linkercomprises 6, 8, 10, 12, 14, 16, or 18 chain atoms (e.g., C, O, N, andS). In certain embodiments, the Linker comprises 6, 8, 10, or 12 chainatoms (e.g., C, O, N, and S).

In certain embodiments, the Linker is a carbon chain optionallysubstituted with non-bulky substituents (e.g., oxo, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₃ alkoxy, OH, halogen, NH₂, NH(C₁-C₃ alkyl),N(C₁-C₃ alkyl)₂, and CN). In certain embodiments, the non-bulkysubstitution is located on the chain carbon atom proximal to the Degron(i.e., the carbon atom is separated from the carbon atom to which theDegron is bonded by at least 3, 4, or 5 chain atoms in the Linker).

In certain embodiments, the Linker is of Formula L0:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein

p1 is an integer selected from 0 to 12;

p2 is an integer selected from 0 to 12;

p3 is an integer selected from 1 to 6;

each W is independently absent, CH₂, O, S, NH or NR₅;

Z is absent, CH₂, O, NH or NR₅;

each R₅ is independently C₁-C₃ alkyl; and

Q is absent or —CH₂C(O)NH—,

wherein the Linker is covalently bonded to the Degron with the

next to Q, and covalently bonded to the Targeting Ligand with the

next to Z, and wherein the total number of chain atoms in the Linker isless than 20.

In certain embodiments, the Linker-Targeting Ligand (TL) has thestructure of Formula L1 or L2:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

p1 is an integer selected from 0 to 12;

p2 is an integer selected from 0 to 12;

p3 is an integer selected from 1 to 6;

each W is independently absent, CH₂, O, S, NH or NR₅;

Z is absent, CH₂, O, NH or NR₅;

each R₅ is independently C₁-C₃ alkyl; and

TL is a Targeting Ligand,

wherein the Linker is covalently bonded to the Degron with

In certain embodiments, p1 is an integer selected from 0 to 10.

In certain embodiments, p1 is an integer selected from 2 to 10.

In certain embodiments, p1 is selected from 1, 2, 3, 4, 5, and 6.

In certain embodiments, p1 is selected from 1, 3, and 5.

In certain embodiments, p1 is selected from 1, 2, and 3.

In certain embodiments, p1 is 3.

In certain embodiments, p2 is an integer selected from 0 to 10.

In certain embodiments, p2 is selected from 0, 1, 2, 3, 4, 5, and 6.

In certain embodiments, p2 is an integer selected from 0 and 1.

In certain embodiments, p3 is an integer selected from 1 to 5.

In certain embodiments, p3 is selected from 2, 3, 4, and 5.

In certain embodiments, p3 is selected from 1, 2, and 3.

In certain embodiments, p3 is selected from 2 and 3.

In certain embodiments, at least one W is CH₂.

In certain embodiments, at least one W is O.

In certain embodiments, at least one W is S.

In certain embodiments, at least one W is NH.

In certain embodiments, at least one W is NR₅; and R₅ is C₁-C₃ alkylselected from methyl, ethyl, and propyl.

In certain embodiments, W is O.

In certain embodiments, Z is absent.

In certain embodiments, Z is CH₂.

In certain embodiments, Z is O.

In certain embodiments, Z is NH.

In certain embodiments, Z is NR₅; and R₅ is C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In certain embodiments, Z is part of the Targeting Ligand that is bondedto the Linker, namely, Z is formed from reacting a functional group ofthe Targeting Ligand with the Linker.

In certain embodiments, W is CH₂, and Z is CH₂.

In certain embodiments, W is O, and Z is CH₂.

In certain embodiments, W is CH₂, and Z is O.

In certain embodiments, W is O, and Z is O.

In certain embodiments, the Linker-Targeting Ligand has the structureof:

wherein Z, TL, and p1 are each as described above.

Any one of the Degrons described herein can be covalently bound to anyone of the Linkers described herein.

In certain embodiments, the present application relates to theDegron-Linker (DL) having the following structure:

wherein each of the variables is as described above in Formula D andFormula L0, and a Targeting Ligand is covalently bonded to the DL withthe

next to Z.

In certain embodiments, the present application relates to theDegron-Linker (DL) having the following structure:

wherein each of the variables is as described above in Formula D andFormula L0, and a Targeting Ligand is covalently bonded to the DL withthe

next to Z.

In certain embodiments, the present application relates to theDegron-Linker (DL) intermediates having the following structure:

or an enantiomer, diastereomer, or stereoisomer thereof,wherein p is 1-19 and X and R₁ are as described above.

In certain embodiments, the DLs have the following structure:

or an enantiomer, diastereomer, or stereoisomer thereof,wherein p is 1-19.

In certain embodiments, the DL intermediate has the following structure:

Some embodiments of the present application relate to a bifunctionalcompound having the following structure:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein each ofthe variables is as described above in Formula D and Formula L0, and theTargeting Ligand is described herein below.

Further embodiments of the present application relate to a bifunctionalcompound having the following structure:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein each ofthe variables is as described above in Formula D and Formula L0, and theTargeting Ligand is described herein below.

Certain embodiments of the present application relate to bifunctionalcompounds having one of the following structures:

In certain embodiments, the Linker may be a polyethylene glycol groupranging in size from about 1 to about 12 ethylene glycol units, between1 and about 10 ethylene glycol units, about 2 about 6 ethylene glycolunits, between about 2 and 5 ethylene glycol units, between about 2 and4 ethylene glycol units.

In certain embodiments, the Linker is designed and optimized based onSAR (structure-activity relationship) and X-ray crystallography of theTargeting Ligand with regard to the location of attachment for theLinker.

In certain embodiments, the optimal Linker length and composition varyby target and can be estimated based upon X-ray structures of theoriginal Targeting Ligand bound to its target. Linker length andcomposition can be also modified to modulate metabolic stability andpharmacokinetic (PK) and pharmacodynamics (PD) parameters.

In certain embodiments, where the Target Ligand binds multiple targets,selectivity may be achieved by varying Linker length where the ligandbinds some of its targets in different binding pockets, e.g., deeper orshallower binding pockets than others.

Targeting Ligand

Targeting Ligand (TL) (or target protein moiety or target protein ligandor ligand) is a small molecule which is capable of binding to or bindsto a target protein of interest.

Some embodiments of the present application relate to TLs which includebut are not limited to Hsp90 inhibitors, kinase inhibitors, MDM2inhibitors, compounds targeting Human BET Bromodomain-containingproteins, compounds targeting cytosolic signaling protein FKBP12, HDACinhibitors, human lysine methyltransferase inhibitors, angiogenesisinhibitors, immunosuppressive compounds, and compounds targeting thearyl hydrocarbon receptor (AHR).

In certain embodiments, the Targeting Ligand is a compound that iscapable of binding to or binds to a kinase, a BET bromodomain-containingprotein, a cytosolic signaling protein (e.g., FKBP12), a nuclearprotein, a histone deacetylase, a lysine methyltransferase, a proteinregulating angiogenesis, a protein regulating immune response, an arylhydrocarbon receptor (AHR), an estrogen receptor, an androgen receptor,a glucocorticoid receptor, or a transcription factor (e.g., SMARCA4,SMARCA2, TRIM24).

In certain embodiments, a kinase to which the Targeting Ligand iscapable of binding or binds includes, but is not limited to, a tyrosinekinase (e.g., AATK, ABL, ABL2, ALK, AXL, BLK, BMX, BTK, CSF1R, CSK,DDR1, DDR2, EGFR, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7,EPHA8, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, ERBB2, ERBB3, ERBB4,FER, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT3, FLT4, FRK, FYN,GSG2, HCK, IGF1R, ILK, INSR, INSRR, IRAK4, ITK, JAK1, JAK2, JAK3, KDR,KIT, KSR1, LCK, LMTK2, LMTK3, LTK, LYN, MATK, MERTK, MET, MLTK, MST1R,MUSK, NPR1, NTRK1, NTRK2, NTRK3, PDGFRA, PDGFRB, PLK4, PTK2, PTK2B,PTK6, PTK7, RET, ROR1, ROR2, ROS1, RYK, SGK493, SRC, SRMS, STYK1, SYK,TEC, TEK, TEX14, TIE1, TNK1, TNK2, TNNI3K, TXK, TYK2, TYRO3, YES1, orZAP70), a serine/threonine kinase (e.g., casein kinase 2, protein kinaseA, protein kinase B, protein kinase C, Raf kinases, CaM kinases, AKT1,AKT2, AKT3, ALK1, ALK2, ALK3, ALK4, Aurora A, Aurora B, Aurora C, CHK1,CHK2, CLK1, CLK2, CLK3, DAPK1, DAPK2, DAPK3, DMPK, ERK1, ERK2, ERK5,GCK, GSK3, HIPK, KHS1, LKB1, LOK, MAPKAPK2, MAPKAPK, MNK1, MSSK1, MST1,MST2, MST4, NDR, NEK2, NEK3, NEK6, NEK7, NEK9, NEK11, PAK1, PAK2, PAK3,PAK4, PAK5, PAK6, PIM1, PIM2, PLK1, RIP2, RIP5, RSK1, RSK2, SGK2, SGK3,SIK1, STK33, TAO1, TAO2, TGF-beta, TLK2, TSSK1, TSSK2, ULK1, or ULK2), acyclin dependent kinase (e.g., Cdk1-Cdk11), and a leucine-rich repeatkinase (e.g., LRRK2).

In certain embodiments, a BET bromodomain-containing protein to whichthe Targeting Ligand is capable of binding or binds includes, but is notlimited to, BRD1, BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, BRD9, BRD10,and BRDT. In certain embodiments, a BET bromodomain-containing proteinis BRD4.

In certain embodiments, a nuclear protein to which the Targeting Ligandis capable of binding or binds includes, but is not limited to, BRD2,BRD3, BRD4, Antennapedia Homeodomain Protein, BRCA1, BRCA2,CCAAT-Enhanced-Binding Proteins, histones, Polycomb-group proteins, HighMobility Group Proteins, Telomere Binding Proteins, FANCA, FANCD2,FANCE, FANCF, hepatocyte nuclear factors, Mad2, NF-kappa B, NuclearReceptor Coactivators, CREB-binding protein, p55, p107, p130, Rbproteins, p53, c-fos, c-jun, c-mdm2, c-myc, and c-rel.

In certain embodiments, the Targeting Ligand is selected from a kinaseinhibitor, a BET bromodomain-containing protein inhibitor, cytosolicsignaling protein FKBP12 ligand, an HDAC inhibitor, a lysinemethyltransferase inhibitor, an angiogenesis inhibitor, animmunosuppressive compound, and an aryl hydrocarbon receptor (AHR)inhibitor.

Non-limiting examples of TLs are shown in below and represent TargetingLigands of certain types of proteins of interest.

In certain embodiments, the present application relates to the compoundscontaining the TL moieties shown in Table 1.

TABLE 1 Targeting Ligands 1-6 Compound Structure TL1

TL2

TL3

TL4

TL5

TL6

TL7

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-I:

or a pharmaceutically acceptable salt thereof, wherein:

is

A₁ is S or C═C;

A₂ is NRa₅ or O;

nn1 is 0, 1, or 2;

each Ra₁ is independently C₁-C₃ alkyl, (CH₂)₀₋₃—CN, (CH₂)₀₋₃-halogen,(CH₂)₀₋₃—OH, (CH₂)₀₋₃—C₁-C₃ alkoxy, C(O)NRa₅L, OL, NRa₅L, or L;

Ra₂ is H, C₁-C₆ alkyl, (CH₂)₀₋₃-heterocyclyl, (CH₂)₀₋₃-phenyl, or L,wherein the heterocyclyl comprises one saturated 5- or 6-membered ringand 1-2 heteroatoms selected from N, O, and S and is optionallysubstituted with C₁-C₃ alkyl, L, or C(O)L, and wherein the phenyl isoptionally substituted with C₁-C₃ alkyl, CN, halogen, OH, C₁-C₃ alkoxy,or L;

nn2 is 0, 1, 2, or 3;

each Ra₃ is independently C₁-C₃ alkyl, (CH₂)₀₋₃—CN, (CH₂)₀₋₃-halogen, L,or C(O)NRa₅L;

Ra₄ is C₁-C₃ alkyl;

Ra₅ is H or C₁-C₃ alkyl; and

L is a Linker,

provided that the compound of Formula TL-I is substituted with only oneL.

In certain embodiments,

is

In certain embodiments,

is

In certain embodiments, A₁ is S.

In certain embodiments, A₁ is C═C.

In certain embodiments, A₂ is NRa₅. In further embodiments, Ra₅ is H. Inother embodiments, Ra₅ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl, ori-propyl). In further embodiments, Ra₅ is methyl.

In certain embodiments, A₂ is O.

In certain embodiments, nn1 is 0.

In certain embodiments, nn1 is 1.

In certain embodiments, nn1 is 2.

In certain embodiments, at least one Ra₁ is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl, or i-propyl). In further embodiments, at least one Ra₁ ismethyl. In further embodiments, two Ra₁ are methyl.

In certain embodiments, at least one Ra₁ is CN, (CH₂)—CN, (CH₂)₂—CN, or(CH₂)₃—CN. In further embodiments, at least one Ra₁ is (CH₂)—CN.

In certain embodiments, at least one Ra₁ is halogen (e.g., F, Cl, orBr), (CH₂)-halogen, (CH₂)₂-halogen, or (CH₂)₃-halogen. In furtherembodiments, at least one Ra₁ is Cl, (CH₂)—Cl, (CH₂)₂—Cl, or (CH₂)₃—Cl.

In certain embodiments, at least one Ra₁ is OH, (CH₂)—OH, (CH₂)₂—OH, or(CH₂)₃—OH.

In certain embodiments, at least one Ra₁ is C₁-C₃ alkoxy (e.g., methoxy,ethoxy, or propoxy), (CH₂)—C₁-C₃ alkoxy, (CH₂)₂—C₁-C₃ alkoxy, or(CH₂)₃—C₁-C₃ alkoxy. In certain embodiments, at least one Ra₁ ismethoxy.

In certain embodiments, one Ra₁ is C(O)NRa₅L. In further embodiments,Ra₅ is H. In other embodiments, Ra₅ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, one Ra₁ is OL.

In certain embodiments, one Ra₁ is NRa₅L. In further embodiments, Ra₅ isH. In other embodiments, Ra₅ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl). In other embodiments, Ra₅ is methyl.

In certain embodiments, one Ra₁ is L.

In certain embodiments, Ra₂ is H.

In certain embodiments, Ra₂ is straight-chain C₁-C₆ or branched C₃-C₆alkyl (e.g., methyl, ethyl, propyl, i-propyl, butyl, i-butyl, t-butyl,pentyl, or hexyl). In further embodiments, Ra₂ is methyl, ethyl, ort-butyl.

In certain embodiments, Ra₂ is heterocyclyl, (CH₂)-heterocyclyl,(CH₂)₂-heterocyclyl, or (CH₂)₃-heterocyclyl. In further embodiments, Ra₂is (CH₂)₃-heterocyclyl. In further embodiments, the heterocyclyl isselected from pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl,isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl,piperazinyl, hexahydropyrimidinyl, morpholinyl, and thiomorpholinyl. Infurther embodiments, the heterocyclyl is piperazinyl.

In certain embodiments, the heterocyclyl is substituted with C₁-C₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl).

In certain embodiments, the heterocyclyl is substituted with C(O)L.

In certain embodiments, the heterocyclyl is substituted with L.

In certain embodiments, Ra₂ is phenyl, (CH₂)-phenyl, (CH₂)₂-phenyl, or(CH₂)₃-phenyl.

In further embodiments, Ra₂ is phenyl.

In certain embodiments, the phenyl is substituted with C₁-C₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl). In certain embodiments, thephenyl is substituted with CN. In certain embodiments, the phenyl issubstituted with halogen (e.g., F, Cl, or Br). In certain embodiments,the phenyl is substituted with OH. In certain embodiments, the phenyl issubstituted with C₁-C₃ alkoxy (e.g., methoxy, ethoxy, or propoxy).

In certain embodiments, the phenyl is substituted with L.

In certain embodiments, Ra₂ is L.

In certain embodiments, nn2 is 0.

In certain embodiments, nn2 is 1.

In certain embodiments, nn2 is 2.

In certain embodiments, nn2 is 3.

In certain embodiments, at least one Ra₃ is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl, or i-propyl). In further embodiments, at least one Ra₃ ismethyl.

In certain embodiments, at least one Ra₃ is CN, (CH₂)—CN, (CH₂)₂—CN, or(CH₂)₃—CN. In further embodiments, at least one Ra₃ is CN.

In certain embodiments, at least one Ra₃ is halogen (e.g., F, Cl, orBr), (CH₂)-halogen, (CH₂)₂-halogen, or (CH₂)₃-halogen. In furtherembodiments, at least one Ra₃ is Cl, (CH₂)—Cl, (CH₂)₂—Cl, or (CH₂)₃—Cl.In further embodiments, at least one Ra₃ is Cl.

In certain embodiments, one Ra₃ is L.

In certain embodiments, one Ra₃ is C(O)NRa₅L. In further embodiments,Ra₅ is H. In other embodiments, Ra₅ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Ra₄ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In further embodiments, Ra₄ is methyl.

In certain embodiments, Ra₅ is H.

In certain embodiments, Ra₅ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In further embodiments, Ra₅ is methyl.

Each of the moieties defined for one of T₁, T₂, T₃, T₄, T₅, A₁, A₂, Ra₁,Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2, can be combined with any of themoieties defined for the others of T₁, T₂, T₃, T₄, T₅, A₁, A₂, Ra₁, Ra₂,Ra₃, Ra₄, Ra₅, nn1, and nn2.

In certain embodiments,

is

and A₁ is S.

In certain embodiments,

is

and A₁ is C═C.

In certain embodiments,

is

and A₁ is C═C.

In certain embodiments, A₂ is NH, and Ra₂ is (CH₂)₀₋₃-heterocyclyl. Infurther embodiments, Ra₂ is (CH₂)₃-heterocyclyl. In further embodiments,the heterocyclyl is piperazinyl. In further embodiments, theheterocyclyl is substituted with C₁-C₃ alkyl, L, or C(O)L.

In certain embodiments, A₂ is NH, and Ra₂ is (CH₂)₀₋₃-phenyl. In furtherembodiments, Ra₂ is phenyl. In further embodiments, the phenyl issubstituted with OH or L.

In certain embodiments, A₂ is NH, and Ra₂ is L.

In certain embodiments, A₂ is NH, and Ra₂ is H or C₁-C₆ alkyl. Infurther embodiments, Ra₂ is C₁-C₄ alkyl.

In certain embodiments, A₂ is O, and Ra₂ is H or C₁-C₆ alkyl. In furtherembodiments, Ra₂ is C₁-C₄ alkyl.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-I1:

or a pharmaceutically acceptable salt thereof, wherein A₂, Ra₁, Ra₂,Ra₃, Ra₄, Ra₅, nn1, and nn2 are each as defined above in Formula TL-I.

Each of A₂, Ra₁, Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2 may be selected fromthe moieties described above in Formula TL-I. Each of the moietiesdefined for one of A₂, Ra₁, Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2, can becombined with any of the moieties defined for the others of A₂, Ra₁,Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2, as described above in Formula TL-I.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-I1a-TL-I1d:

or a pharmaceutically acceptable salt thereof, wherein:

each Ra₆ is independently C₁-C₃ alkyl, (CH₂)₀₋₃—CN, (CH₂)₀₋₃-halogen,(CH₂)₀₋₃—OH, or (CH₂)₀₋₃—C₁-C₃ alkoxy;

Ra₇ is (CH₂)₀₋₃-heterocyclyl, (CH₂)₀₋₃-phenyl, or L, wherein theheterocyclyl comprises one saturated 5- or 6-membered ring and 1-2heteroatoms selected from N, O, and S and is substituted with L orC(O)L, and wherein the phenyl is substituted with L;

Ra₈ is H, C₁-C₆ alkyl, (CH₂)₀₋₃-heterocyclyl, or (CH₂)₀₋₃-phenyl,wherein the heterocyclyl comprises one saturated 5- or 6-membered ringand 1-2 heteroatoms selected from N, O, and S and is optionallysubstituted with C₁-C₃ alkyl, and wherein the phenyl is optionallysubstituted with C₁-C₃ alkyl, CN, halogen, OH, or C₁-C₃ alkoxy;

Ra₁₀ is C₁-C₃ alkyl, (CH₂)₀₋₃—CN, or (CH₂)₀₋₃-halogen; and

A₂, Ra₄, Ra₅, nn1, and L are each as defined above in Formula TL-I.

In certain embodiments, nn1 is 0.

In certain embodiments, nn1 is 1.

In certain embodiments, nn1 is 2.

In certain embodiments, at least one Ra₆ is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl, or i-propyl). In further embodiments, at least one Ra₆ ismethyl. In further embodiments, two Ra₆ are methyl.

In certain embodiments, at least one Ra₆ is CN, (CH₂)—CN, (CH₂)₂—CN, or(CH₂)₃—CN. In further embodiments, at least one Ra₆ is (CH₂)—CN.

In certain embodiments, at least one Ra₆ is halogen (e.g., F, Cl, orBr), (CH₂)-halogen, (CH₂)₂-halogen, or (CH₂)₃-halogen. In furtherembodiments, at least one Ra₆ is Cl, (CH₂)—Cl, (CH₂)₂—Cl, or (CH₂)₃—Cl.

In certain embodiments, at least one Ra₆ is OH, (CH₂)—OH, (CH₂)₂—OH, or(CH₂)₃—OH.

In certain embodiments, at least one Ra₆ is C₁-C₃ alkoxy (e.g., methoxy,ethoxy, or propoxy), (CH₂)—C₁-C₃ alkoxy, (CH₂)₂—C₁-C₃ alkoxy, or(CH₂)₃—C₁-C₃ alkoxy. In certain embodiments, at least one Ra₆ ismethoxy.

In certain embodiments, Ra₇ is heterocyclyl, (CH₂)-heterocyclyl,(CH₂)₂-heterocyclyl, or (CH₂)₃-heterocyclyl. In further embodiments, Ra₇is (CH₂)₃-heterocyclyl. In further embodiments, the heterocyclyl isselected from pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl,isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl,piperazinyl, hexahydropyrimidinyl, morpholinyl, and thiomorpholinyl. Infurther embodiments, the heterocyclyl is piperazinyl.

In certain embodiments, the heterocyclyl is substituted with C(O)L.

In certain embodiments, the heterocyclyl is substituted with L.

In certain embodiments, Ra₇ is phenyl, (CH₂)-phenyl, (CH₂)₂-phenyl, or(CH₂)₃-phenyl. In further embodiments, Ra₇ is phenyl.

In certain embodiments, Ra₇ is L.

In certain embodiments, Ra₈ is H.

In certain embodiments, Ra₈ is straight-chain C₁-C₆ or branched C₃-C₆alkyl (e.g., methyl, ethyl, propyl, i-propyl, butyl, i-butyl, t-butyl,pentyl, or hexyl). In further embodiments, Ra₈ is methyl, ethyl, ort-butyl.

In certain embodiments, Ra₈ is heterocyclyl, (CH₂)-heterocyclyl,(CH₂)₂-heterocyclyl, or (CH₂)₃-heterocyclyl. In further embodiments, Ra₈is (CH₂)₃-heterocyclyl. In further embodiments, the heterocyclyl isselected from pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl,isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl,piperazinyl, hexahydropyrimidinyl, morpholinyl, and thiomorpholinyl. Infurther embodiments, the heterocyclyl is piperazinyl.

In certain embodiments, the heterocyclyl is substituted with C₁-C₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl).

In certain embodiments, Ra₈ is phenyl, (CH₂)-phenyl, (CH₂)₂-phenyl, or(CH₂)₃-phenyl. In further embodiments, Ra₈ is phenyl.

In certain embodiments, the phenyl is substituted with C₁-C₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl). In certain embodiments, thephenyl is substituted with CN. In certain embodiments, the phenyl issubstituted with halogen (e.g., F, Cl, or Br). In certain embodiments,the phenyl is substituted with OH. In certain embodiments, the phenyl issubstituted with C₁-C₃ alkoxy (e.g., methoxy, ethoxy, or propoxy).

In certain embodiments, Ra₁₀ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Ra₁₀ is CN, (CH₂)—CN, (CH₂)₂—CN, or (CH₂)₃—CN.

In certain embodiments, Ra₁₀ is halogen (e.g., F, Cl, or Br),(CH₂)-halogen, (CH₂)₂-halogen, or (CH₂)₃-halogen. In furtherembodiments, Ra₁₀ is Cl, (CH₂)—Cl, (CH₂)₂—Cl, or (CH₂)₃—Cl. In furtherembodiments, Ra₁₀ is Cl.

Each of A₂, Ra₄, Ra₅, and nn1 may be selected from the moietiesdescribed above in Formula TL-I. Each of the moieties defined for one ofA₂, Ra₄, Ra₅, Ra₆, Ra₇, Ra₈, Ra₁₀, and nn1, can be combined with any ofthe moieties defined for the others of A₂, Ra₄, Ra₅, Ra₆, Ra₇, Ra₈,Ra₁₀, and nn1, as described above and in Formula TL-I.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-I2:

or a pharmaceutically acceptable salt thereof, wherein A₂, Ra₁, Ra₂,Ra₃, Ra₄, R_(a5), nn1, and nn2 are each as defined above in FormulaTL-I.

Each of A₂, Ra₁, Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2 may be selected fromthe moieties described above in Formula TL-I. Each of the moietiesdefined for one of A₂, Ra₁, Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2, can becombined with any of the moieties defined for the others of A₂, Ra₁,Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2, as described above in Formula TL-I.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-12a-TL-12c:

or a pharmaceutically acceptable salt thereof, wherein A₂, Ra₄, Ra₅,nn1, and L are each as defined above in Formula TL-I, and Ra₆, R_(a7),R_(a8), and Ra₁₀ are each as defined above in Formula TL-I1a-TL-I1d.

Each of A₂, Ra₄, Ra₅, and nn1 may be selected from the moietiesdescribed above in Formula TL-I, and each of Ra₆, Ra₇, Ra₈, and Ra₁₀ maybe selected from the moieties described above in Formula TL-I1a-TL-I1d.Each of the moieties defined for one of A₂, Ra₄, Ra₅, Ra₆, Ra₇, Ra₈,Ra₁₀, and nn1, can be combined with any of the moieties defined for theothers of A₂, Ra₄, Ra₅, Ra₆, Ra₇, Ra₈, Ra₁₀, and nn1, as described abovein Formula TL-I and TL-I1a-TL-I1d.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-I3:

or a pharmaceutically acceptable salt thereof.

A₂, Ra₁, Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2 are each as defined above inFormula TL-I. Each of A₂, Ra₁, Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2 may beselected from the moieties described above in Formula TL-I. Each of themoieties defined for one of A₂, Ra₁, Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2,can be combined with any of the moieties defined for the others of A₂,Ra₁, Ra₂, Ra₃, Ra₄, Ra₅, nn1, and nn2, as described above in FormulaTL-I.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-13a-TL-13c:

or a pharmaceutically acceptable salt thereof, wherein:

Ra₉ is C(O)NRa₅L, OL, NRa₅L, or L;

A₂, Ra₄, Ra₅, nn1, and L are each as defined above in Formula TL-I; and

Ra₆, Ra₇, Ra₈, and Ra₁₀ are each as defined above in FormulaTL-I1a-TL-I1d.

In certain embodiments, Ra₉ is C(O)NRa₅L. In further embodiments, Ra₅ isH. In other embodiments, Ra₅ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Ra₉ is OL.

In certain embodiments, Ra₉ is NRa₅L. In further embodiments, Ra₅ is H.In other embodiments, Ra₅ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In other embodiments, Ra₅ is methyl.

In certain embodiments, Ra₉ is L.

Each of A₂, Ra₄, Ra₅, and nn1 may be selected from the moietiesdescribed above in Formula TL-I, and each of Ra₆, Ra₇, Ra₈, and Ra₁₀ maybe selected from the moieties described above in Formula TL-I1a-TL-I1d.Each of the moieties defined for one of A₂, Ra₄, Ra₅, Ra₆, Ra₇, Ra₈,Ra₉, Ra₁₀, and nn1, can be combined with any of the moieties defined forthe others of A₂, Ra₄, Ra₅, Ra₆, Ra₇, Ra₈, Ra₉, Ra₁₀, and nn1, asdescribed above and in Formula TL-I and TL-I1a-TL-I1d.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-II:

or a pharmaceutically acceptable salt thereof, wherein:

T₆ is CRb₄ or N;

Rb₁, Rb₂, and Rb₅ are each independently H or C₁-C₃ alkyl;

Rb₃ is C₃-C₆ cycloalkyl;

each Rb₄ is independently H, C₁-C₃ alkyl, C₁-C₃ alkoxy, CN, or halogen;

nn3 is 0, 1, 2, or 3;

each Rb₆ is independently C₁-C₃ alkyl, C₁-C₃ alkoxy, CN, or halogen;

Rb₇ is C(O)NRb₈L, OL, NRb₈L, or L;

Rb₈ is H or C₁-C₃ alkyl; and

L is a Linker.

In certain embodiments, T₆ is CRb₄.

In certain embodiments, T₆ is N.

In certain embodiments, Rb₁ is H. In certain embodiments, Rb₁ is C₁-C₃alkyl (e.g., methyl, ethyl, propyl, or i-propyl). In furtherembodiments, Rb₁ is methyl.

In certain embodiments, Rb₂ is H. In certain embodiments, Rb₂ is C₁-C₃alkyl (e.g., methyl, ethyl, propyl, or i-propyl). In furtherembodiments, Rb₂ is methyl or ethyl.

In certain embodiments, Rb₅ is H. In certain embodiments, Rb₅ is C₁-C₃alkyl (e.g., methyl, ethyl, propyl, or i-propyl).

In certain embodiments, Rb₃ is cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl. In further embodiments, Rb₃ is cyclopentyl.

In certain embodiments, Rb₄ is H.

In certain embodiments, Rb₄ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl).

In certain embodiments, Rb₄ is C₁-C₃ alkoxy (e.g., methoxy, ethoxy, orpropoxy).

In certain embodiments, Rb₄ is CN.

In certain embodiments, Rb₄ is halogen (e.g., F, Cl, or Br).

In certain embodiments, nn3 is 0.

In certain embodiments, nn3 is 1.

In certain embodiments, nn3 is 2.

In certain embodiments, nn3 is 3.

In certain embodiments, at least one Rb₆ is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl, or i-propyl). In further embodiments, at least one Rb₆ ismethyl.

In certain embodiments, at least one Rb₆ is C₁-C₃ alkoxy (e.g., methoxy,ethoxy, or propoxy). In further embodiments, at least one Rb₆ ismethoxy.

In certain embodiments, at least one Rb₆ is CN.

In certain embodiments, at least one Rb₆ is halogen (e.g., F, Cl, orBr).

In certain embodiments, Rb₇ is C(O)NRb₈L. In further embodiments, Rb₈ isH. In other embodiments, Rb₈ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Rb₇ is OL.

In certain embodiments, Rb₇ is NRb₈L. In further embodiments, Rb₈ is H.In other embodiments, Rb₈ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In other embodiments, Rb₈ is methyl.

In certain embodiments, Rb₇ is L.

Each of the moieties defined for one of T₆, Rb₁, Rb₂, Rb₃, Rb₄, Rb₅,Rb₆, Rb₇, Rb₈, and nn3, can be combined with any of the moieties definedfor the others of T₆, Rb₁, Rb₂, Rb₃, Rb₄, Rb₅, Rb₆, Rb₇, Rb₈, and nn3.

In certain embodiments, Rb₃ is cyclopentyl, and Rb₇ is C(O)NRb₈L.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-II1:

or a pharmaceutically acceptable salt thereof, wherein T₆, Rb₂, Rb₄,Rb₆, Rb₇, and Rb₈ are each as defined above in Formula TL-II.

Each of T₆, Rb₂, Rb₄, Rb₆, Rb₇, and Rb₈ may be selected from themoieties described above in Formula TL-II. Each of the moieties definedfor one of T₆, Rb₂, Rb₄, Rb₆, Rb₇, and Rb₈, can be combined with any ofthe moieties defined for the others of T₆, Rb₂, Rb₄, Rb₆, Rb₇, and Rb₈,as described above in Formula TL-II.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-II1a:

or a pharmaceutically acceptable salt thereof, wherein T₆, Rb₂, and Rb₄are each as defined above in Formula TL-II.

Each of T₆, Rb₂, and Rb₄ may be selected from the moieties describedabove in Formula TL-II. Each of the moieties defined for one of T₆, Rb₂,and Rb₄, can be combined with any of the moieties defined for the othersof T₆, Rb₂, and Rb₄, as described above in Formula TL-II.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-III:

or a pharmaceutically acceptable salt thereof, wherein:

nn4 is 0 or 1;

Rc₁ is C(O)NRc₆L, OL, NRc₆L, or L;

Rc₂ is H, C₁-C₃ alkyl, C(O)NRc₆L, OL, NRc₆L, or L;

Rc₃ is H, C₁-C₃ alkyl, C(O)L, or L;

nn5 is 0, 1, or 2;

each Rc₄ is independently C₁-C₃ alkyl or C₁-C₃ alkoxy;

each Rc₅ is independently H or C₁-C₃ alkyl;

Rc₆ is independently H or C₁-C₃ alkyl; and

L is a Linker, provided that the compound of Formula TL-III issubstituted with only one L.

In certain embodiments, nn4 is 0.

In certain embodiments, nn4 is 1.

In certain embodiments, Rc₁ is C(O)NRc₆L. In further embodiments, Rc₆ isH. In other embodiments, Rc₆ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Rc₁ is OL.

In certain embodiments, Rc₁ is NRc₆L. In further embodiments, Rc₆ is H.In other embodiments, Rc₆ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In other embodiments, Rc₆ is methyl.

In certain embodiments, Rc₁ is L.

In certain embodiments, Rc₂ is H.

In certain embodiments, Rc₂ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In further embodiments, Rc₂ is methyl.

In certain embodiments, Rc₂ is C(O)NRc₆L. In further embodiments, Rc₆ isH. In other embodiments, Rc₆ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Rc₂ is OL.

In certain embodiments, Rc₂ is NRc₆L. In further embodiments, Rc₆ is H.In other embodiments, Rc₆ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In other embodiments, Rc₆ is methyl.

In certain embodiments, Rc₂ is L.

In certain embodiments, Rc₃ is H.

In certain embodiments, Rc₃ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl).

In certain embodiments, Rc₃ is C(O)L.

In certain embodiments, Rc₃ is L.

In certain embodiments, nn5 is 0.

In certain embodiments, nn5 is 1.

In certain embodiments, nn5 is 2.

In certain embodiments, at least one Rc₄ is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl, or i-propyl). In further embodiments, at least one Rc₄ ismethyl.

In certain embodiments, at least one Rc₄ is C₁-C₃ alkoxy (e.g., methoxy,ethoxy, or propoxy). In further embodiments, at least one Rc₄ ismethoxy.

In certain embodiments, at least one Rc₅ is H.

In certain embodiments, at least one Rc₅ is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl, or i-propyl). In further embodiments, at least one Rc₅ ismethyl. In further embodiments, two Rc₅ are methyl.

Each of the moieties defined for one of Rc₁, Rc₂, Rc₃, Rc₄, Rc₅, Rc₆,nn4, and nn5, can be combined with any of the moieties defined for theothers of Rc₁, Rc₂, Rc₃, Rc₄, Rc₅, Rc₆, nn4, and nn5.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-III1-TL-III3:

or a pharmaceutically acceptable salt thereof, wherein Rc₁, Rc₂, Rc₃,Rc₄, and nn5 are each as defined above in Formula TL-III.

Each of Rc₁, Rc₂, Rc₃, Rc₄, and nn5 may be selected from the moietiesdescribed above in Formula TL-III. Each of the moieties defined for oneof Rc₁, Rc₂, Rc₃, Rc₄, and nn5, can be combined with any of the moietiesdefined for the others of Rc₁, Rc₂, Rc₃, Rc₄, and nn5, as describedabove in Formula TL-III.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-IV:

or a pharmaceutically acceptable salt thereof, wherein:

each Rd₁ is independently H or C₁-C₃ alkyl;

nn6 is 0, 1, 2, or 3;

nn7 is 0, 1, 2, or 3;

each Rd₂ is independently C₁-C₃ alkyl, C₁-C₃ alkoxy, CN, or halogen;

Rd₃ is C(O)NRd₄L, OL, NRd₄L, or L;

Rd₄ is H or C₁-C₃ alkyl; and

L is a Linker.

In certain embodiments, Rd₁ is H.

In certain embodiments, Rd₁ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In further embodiments, Rd₁ is methyl.

In certain embodiments, nn6 is 0.

In certain embodiments, nn6 is 1.

In certain embodiments, nn6 is 2.

In certain embodiments, nn6 is 3.

In certain embodiments, nn7 is 0.

In certain embodiments, nn7 is 1.

In certain embodiments, nn7 is 2.

In certain embodiments, nn7 is 3.

In certain embodiments, at least one Rd₂ is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl, or i-propyl). In further embodiments, at least one Rd₂ ismethyl.

In certain embodiments, at least one Rd₂ is C₁-C₃ alkoxy (e.g., methoxy,ethoxy, or propoxy). In further embodiments, at least one Rd₂ ismethoxy.

In certain embodiments, at least one Rd₂ is CN.

In certain embodiments, at least one Rd₂ is halogen (e.g., F, Cl, orBr).

In certain embodiments, Rd₃ is C(O)NRd₄L. In further embodiments, Rd₄ isH. In other embodiments, Rd₄ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Rd₃ is OL.

In certain embodiments, Rd₃ is NRd₄L. In further embodiments, Rd₄ is H.In other embodiments, Rd₄ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In other embodiments, Rd₄ is methyl.

In certain embodiments, Rd₃ is L.

Each of the moieties defined for one of Rd₁, Rd₂, Rd₃, Rd₄, nn6, andnn7, can be combined with any of the moieties defined for the others ofRd₁, Rd₂, Rd₃, Rd₄, nn6, and nn7.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-IV1:

or a pharmaceutically acceptable salt thereof, wherein Rd₃ is as definedabove in Formula TL-IV. Rd₃ may be selected from the moieties describedabove in Formula TL-IV.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-V:

or a pharmaceutically acceptable salt thereof, wherein:

each Re₁ is independently H or C₁-C₃ alkyl;

nn8 is 0, 1, 2, or 3;

nn9 is 0, 1, 2, or 3;

each Re₂ is independently C₁-C₃ alkyl, C₁-C₃ alkoxy, CN, or halogen;

Re₃ is NH—(CH₂)₁₋₃—C(O)NRe₄L, C(O)NRe₄L, OL, NRe₄L, or L;

Re₄ is H or C₁-C₃ alkyl; and

L is a Linker.

In certain embodiments, Re₁ is H.

In certain embodiments, Re₁ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In further embodiments, Re₁ is methyl.

In certain embodiments, nn8 is 0.

In certain embodiments, nn8 is 1.

In certain embodiments, nn8 is 2.

In certain embodiments, nn8 is 3.

In certain embodiments, nn9 is 0.

In certain embodiments, nn9 is 1.

In certain embodiments, nn9 is 2.

In certain embodiments, nn9 is 3.

In certain embodiments, at least one Re₂ is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl, or i-propyl). In further embodiments, at least one Re₂ ismethyl.

In certain embodiments, at least one Re₂ is C₁-C₃ alkoxy (e.g., methoxy,ethoxy, or 10 propoxy). In further embodiments, at least one Re₂ ismethoxy.

In certain embodiments, at least one Re₂ is CN.

In certain embodiments, at least one Re₂ is halogen (e.g., F, Cl, orBr).

In certain embodiments, Re₃ is NH—CH₂—C(O)NRe₄L, NH—(CH₂)₂—C(O)NRe₄L, orNH—(CH₂)₃—C(O)NRe₄L. In further embodiments, Re₃ is NH—CH₂—C(O)NRe₄L. Infurther embodiments, Re₄ is H. In other embodiments, Re₄ is C₁-C₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl).

In certain embodiments, Re₃ is C(O)NRe₄L. In further embodiments, Re₄ isH. In other embodiments, Re₄ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Re₃ is OL.

In certain embodiments, Re₃ is NRe₄L. In further embodiments, Re₄ is H.In other embodiments, Re₄ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In other embodiments, Re₄ is methyl.

In certain embodiments, Re₃ is L.

Each of the moieties defined for one of Re₁, Re₂, Re₃, Re₄, nn8, andnn9, can be combined with any of the moieties defined for the others ofRe₁, Re₂, Re₃, Re₄, nn8, and nn9.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-V1:

or a pharmaceutically acceptable salt thereof, wherein Re₃ is as definedabove in Formula TL-V. Re₃ may be selected from the moieties describedabove in Formula TL-V.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-VI:

or a pharmaceutically acceptable salt thereof, wherein:

Rf₁ is C(O)NRf₂L, OL, NRf₂L, or L;

Rf₂ is independently H or C₁-C₃ alkyl; and

L is a Linker.

In certain embodiments, Rf₁ is C(O)NRf₂L. In further embodiments, Rf₂ isH. In other embodiments, Rf₂ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Rf₁ is OL.

In certain embodiments, Rf₁ is NRe₄L. In further embodiments, Rf₂ is H.In other embodiments, Rf₂ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In other embodiments, Rf₂ is methyl.

In certain embodiments, Rf₁ is L.

In certain embodiments, a Targeting Ligand is a compound of FormulaTL-VII:

or a pharmaceutically acceptable salt thereof, wherein:

T₇ is CH₂ or CH₂CH₂;

Rg₁ is C(O)Rg₅ or (CH₂)₁₋₃Rg₆;

-   -   nn10 is 0, 1, 2, or 3;    -   nn11 is 0, 1, 2, or 3;    -   each Rg₂ is independently C₁-C₃ alkyl, C₁-C₃ alkoxy, CN, or        halogen;

Rg₃ is C(O)NRg₄L, OL, NRg₄L, L, O—(CH₂)₁₋₃—C(O)NRg₄L, orNHC(O)—(CH₂)₁₋₃—C(O)NRg₄L;

Rg₄ is H or C₁-C₃ alkyl;

Rg₅ is C₁-C₆ alkyl;

Rg₆ is phenyl optionally substituted with C₁-C₃ alkyl, C₁-C₃ alkoxy, CN,or halogen; and

L is a Linker.

In certain embodiments, T₇ is CH₂.

In certain embodiments, T₇ is CH₂CH₂.

In certain embodiments, Rg₁ is C(O)Rg₅.

In certain embodiments, Rg₁ is (CH₂)-Rg₆, (CH₂)₂-Rg₆, or (CH₂)₃-Rg₆.

In certain embodiments, Rg₅ is straight-chain C₁-C₆ or branched C₃-C₆alkyl (e.g., methyl, ethyl, propyl, i-propyl, butyl, i-butyl, t-butyl,pentyl, or hexyl).

In certain embodiments, Rg₆ is unsubstituted phenyl.

In certain embodiments, Rg₆ is phenyl substituted with one, two, three,or more substituents independently selected from C₁-C₃ alkyl (e.g.,methyl, ethyl, propyl, or i-propyl), C₁-C₃ alkoxy (e.g., methoxy,ethoxy, or propoxy), CN, and halogen (e.g., F, Cl, or Br).

In certain embodiments, nn10 is 0.

In certain embodiments, nn10 is 1.

In certain embodiments, nn10 is 2.

In certain embodiments, nn10 is 3.

In certain embodiments, nn11 is 0.

In certain embodiments, nn11 is 1.

In certain embodiments, nn11 is 2.

In certain embodiments, nn11 is 3.

In certain embodiments, at least one Rg₂ is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl, or i-propyl). In further embodiments, at least one Rg₂ ismethyl.

In certain embodiments, at least one Rg₂ is C₁-C₃ alkoxy (e.g., methoxy,ethoxy, or propoxy). In further embodiments, at least one Rg₂ ismethoxy.

In certain embodiments, at least one Rg₂ is CN.

In certain embodiments, at least one Rg₂ is halogen (e.g., F, Cl, orBr).

In certain embodiments, Rg₃ is C(O)NRg₄L. In further embodiments, Rg₄ isH. In other embodiments, Rg₄ is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl, or i-propyl).

In certain embodiments, Rg₃ is OL.

In certain embodiments, Rg₃ is NRg₄L. In further embodiments, Rg₄ is H.In other embodiments, Rg₄ is C₁-C₃ alkyl (e.g., methyl, ethyl, propyl,or i-propyl). In other embodiments, Rg₄ is methyl.

In certain embodiments, Rg₃ is L.

In certain embodiments, Rg₃ is O—(CH₂)—C(O)NRg₄L, O—(CH₂)₂—C(O)NRg₄L, orO—(CH₂)₃—C(O)NRg₄L. In further embodiments, Rg₃ is O—(CH₂)—C(O)NRg₄L. Infurther embodiments, Rg₄ is H. In other embodiments, Rg₄ is C₁-C₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl).

In certain embodiments, Rg₃ is NHC(O)—(CH₂)—C(O)NRg₄L,NHC(O)—(CH₂)₂—C(O)NRg₄L, or NHC(O)—(CH₂)₃—C(O)NRg₄L. In furtherembodiments, Rg₃ is NHC(O)—(CH₂)—C(O)NRg₄L, NHC(O)—(CH₂)₂—C(O)NRg₄L. Infurther embodiments, Rg₃ is NHC(O)—(CH₂)₂—C(O)NRg₄L. In furtherembodiments, Rg₄ is H. In other embodiments, Rg₄ is C₁-C₃ alkyl (e.g.,methyl, ethyl, propyl, or i-propyl).

In certain embodiments, the Targeting Ligand is selected from thefollowing, wherein R is a Linker:

In certain embodiments, the TLs or targets are chosen based on existence(known target protein binding moieties) and ability to develop potentand selective ligands with functional positions that can accommodate aLinker. Some embodiments relate to targets with less selectivity, whichmay benefit from degradation coupled with proteomics as a measure ofcompound selectivity or target ID. Such cases include, but are notlimited to a) targets that have multiple functionalities that are unableto be targeted by inhibition; b) targets that are resistant toinhibitors without altering their binding; c) targets that have ligandsthat do not alter the function of the target; and d) targets that wouldbenefit from irreversible inhibition but lack reactive residues that canbe targeted with covalent ligands.

In certain embodiments, the present application relates to smallmolecule inducers of protein degradation, which have numerous advantagesover inhibitors of protein function and can a) overcome resistance incertain cases; b) prolong the kinetics of drug effect by destroying theprotein requiring resynthesis even after the small molecule has beenmetabolized; c) target all functions of a protein at once rather than aspecific catalytic activity or binding event; d) expand the number ofdrug targets by including all proteins that a ligand can be developedfor, rather than proteins whose activity can be affected by a smallmolecule inhibitor, antagonist or agonist; and e) have increased potencycompared to inhibitors due to the possibility of the small moleculeacting catalytically.

Some embodiments of the present application relate to degradation orloss of 30% to 100% of the target protein. Certain embodiments relate tothe loss of 50-100% of the target protein. Other embodiments relate tothe loss of 75-95% of the targeted protein.

Some embodiments of present application relate to the bifunctionalcompounds having the following structures, their synthesis and methodsof use:

Some of the foregoing compounds can comprise one or more asymmetriccenters, and thus can exist in various isomeric forms, e.g.,stereoisomers and/or diastereomers. Thus, inventive compounds andpharmaceutical compositions thereof may be in the form of an individualenantiomer, diastereomer or geometric isomer, or may be in the form of amixture of stereoisomers. In certain embodiments, the compounds of theapplication are enantiopure compounds. In certain other embodiments,mixtures of stereoisomers or diastereomers are provided.

Furthermore, certain compounds, as described herein may have one or moredouble bonds that can exist as either the Z or E isomer, unlessotherwise indicated. The application additionally encompasses thecompounds as individual isomers substantially free of other isomers andalternatively, as mixtures of various isomers, e.g., racemic mixtures ofstereoisomers. In addition to the above-mentioned compounds per se, thisapplication also encompasses pharmaceutically acceptable derivatives ofthese compounds and compositions comprising one or more compounds of theapplication and one or more pharmaceutically acceptable excipients oradditives.

Compounds of the application may be prepared by crystallization of thecompound under different conditions and may exist as one or acombination of polymorphs of the compound forming part of thisapplication. For example, different polymorphs may be identified and/orprepared using different solvents, or different mixtures of solvents forrecrystallization; by performing crystallizations at differenttemperatures; or by using various modes of cooling, ranging from veryfast to very slow cooling during crystallizations. Polymorphs may alsobe obtained by heating or melting the compound followed by gradual orfast cooling. The presence of polymorphs may be determined by solidprobe NMR spectroscopy, IR spectroscopy, differential scanningcalorimetry, powder X-ray diffractogram and/or other techniques. Thus,the present application encompasses inventive compounds, theirderivatives, their tautomeric forms, their stereoisomers, theirpolymorphs, their pharmaceutically acceptable salts theirpharmaceutically acceptable solvates and pharmaceutically acceptablecompositions containing them.

In certain embodiments, the compounds of the present application areuseful as anticancer agents, and thus may be useful in the treatment ofcancer, by effecting tumor cell death or inhibiting the growth of tumorcells. In certain exemplary embodiments, the disclosed anticancer agentsare useful in the treatment of cancers and other proliferativedisorders, including, but not limited to breast cancer, cervical cancer,colon and rectal cancer, leukemia, lung cancer, melanoma, multiplemyeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer,prostate cancer, gastric cancer, leukemias (e.g., myeloid, lymphocytic,myelocytic and lymphoblastic leukemias), malignant melanomas, T-celllymphoma.

Synthesis of the Compounds of the Application

Exemplary synthetic schemes for preparing the bifunctional compounds ofthe present application are shown in below.

In one aspect of the application, a method for the synthesis of the corestructure of Degron-Linker moiety of certain compounds is provided, themethod comprising steps of:

a) reacting tert-Butyl (2-aminoethyl)carbamate or its analog (e.g.,n=1-20) (1) or its analog (e.g., n=1-20) with chloroacetyl chlorideunder suitable conditions to generate tert-butyl(2-(2-chloroacetamido)ethyl)carbamate or its analog (e.g., n=1-20) (2);

b) reacting tert-butyl (2-(2-chloroacetamido)ethyl)carbamate or itsanalog (2) with dimethyl 3-hydroxyphthalate under suitable conditions toprovide dimethyl3-(2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-oxoethoxy)phthalateor its analog (3);

c) reacting dimethyl3-(2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-oxoethoxy)phthalateor its analog (3) with strong base, followed by3-aminopiperidine-2,6-dione hydrochloride to generate tert-butyl(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethyl)carbamateor its analog (4);

d) deprotecting compound (4) to providediaminoethyl-acetyl-O-thalidomide trifluoroacetate or its analog (5).

Diaminobutyl-acetyl-O-thalidomide trifluoroacetate can be preparedaccording to the procedure in Fischer et al. Nature, 2014, 512, 49-53.

In another aspect of the application, a method for the synthesis of theexemplary bifunctional compound is provided, the method comprisingreacting a Degron-Linker moiety, for example, compound (5) with an acidderivative of a Target Ligand R-compound (6) under suitable conditionsto yield a bifunctional compound (7).

Those of skill in the art will realize that based on this teaching andthose known in the art, one could prepare any of the compounds of thepresent application.

In yet another aspect of the application, methods for producingintermediates useful for the preparation of certain compounds of theapplication are provided.

In some aspects of the application, the Degron-Linker intermediates canbe prepared according to the following steps:

In other aspects of the application, the bifunctional compoundsdBET1-dBET6 can be prepared according to the following schemes using amoiety targeting bromodomain:

In other aspects of the application, the bifunctional compounds dGR1 anddGR2 can be prepared according to the following schemes using TL4 targetmoiety (dexamethasone):

In other embodiments of the disclosure, the bifunctional compoundsdFKBP-1 and dFKBP-2 can be prepared using TL5 target moiety (AP1479)according to the general methods illustrated above, as shown in thefollowing schemes:

Scheme for synthesis of amino acid-thalidomide linker

In certain embodiments, the methods described above are carried out insolution phase. In certain other embodiments, the methods describedabove are carried out on a solid phase. In certain embodiments, thesynthetic method is amenable to high-throughput techniques or totechniques commonly used in combinatorial chemistry.

Pharmaceutical Compositions

Accordingly, in another aspect of the present application,pharmaceutical compositions are provided, which comprise any one of thecompounds described herein (or a prodrug, pharmaceutically acceptablesalt or other pharmaceutically acceptable derivative thereof), andoptionally comprise a pharmaceutically acceptable carrier. In certainembodiments, these compositions optionally further comprise one or moreadditional therapeutic agents. Alternatively, a compound of thisapplication may be administered to a patient in need thereof incombination with the administration of one or more other therapeuticagents. For example, additional therapeutic agents for conjointadministration or inclusion in a pharmaceutical composition with acompound of this application may be, for example, an approvedchemotherapeutic agent, or it may be any one of a number of agentsundergoing approval in the Food and Drug Administration that ultimatelyobtain approval for the treatment of any disorder associated withcellular hyperproliferation. In certain other embodiments, theadditional therapeutic agent is an anticancer agent, as discussed inmore detail herein.

It will also be appreciated that certain of the compounds of presentapplication can exist in free form for treatment, or where appropriate,as a pharmaceutically acceptable derivative thereof. According to thepresent application, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or a pro-drug or other adduct or derivative of acompound of this application which upon administration to a patient inneed is capable of providing, directly or indirectly, a compound asotherwise described herein, or a metabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts of amines, carboxylic acids, and other types ofcompounds, are well known in the art. For example, S. M. Berge, et al.describe pharmaceutically acceptable salts in detail in J PharmaceuticalSciences, 66:1-19 (1977), incorporated herein by reference. The saltscan be prepared in situ during the final isolation and purification ofthe compounds of the application, or separately by reacting a free baseor free acid function with a suitable reagent, as described generallybelow. For example, a free base function can be reacted with a suitableacid. Furthermore, where the compounds of the application carry anacidic moiety, suitable pharmaceutically acceptable salts thereof may,include metal salts such as alkali metal salts, e.g. sodium or potassiumsalts; and alkaline earth metal salts, e.g. calcium or magnesium salts.Examples of pharmaceutically acceptable, nontoxic acid addition saltsare salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters that hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moeity advantageously has not more than 6carbon atoms. Examples of particular esters include formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

Furthermore, the term “pharmaceutically acceptable prodrugs” as usedherein refers to those prodrugs of the compounds of the presentapplication which are, within the scope of sound medical judgment,suitable for use in contact with the issues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the application. The term “prodrug” refers tocompounds that are rapidly transformed in vivo to yield the parentcompound of the above formula, for example by hydrolysis in blood. Athorough discussion is provided in T. Higuchi and V. Stella, Pro-drugsas Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, andin Edward B. Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated herein by reference.

As described above, the pharmaceutical compositions of the presentapplication additionally comprise a pharmaceutically acceptable carrier,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theapplication, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this application. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatine; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil, sesame oil; olive oil; corn oil and soybean oil;glycols; such as propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension orcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionthat, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisapplication with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner.

Examples of embedding compositions that can be used include polymericsubstances and waxes. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose and starch. Such dosage forms may alsocomprise, as in normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

The present application encompasses pharmaceutically acceptable topicalformulations of inventive compounds. The term “pharmaceuticallyacceptable topical formulation”, as used herein, means any formulationwhich is pharmaceutically acceptable for intradermal administration of acompound of the application by application of the formulation to theepidermis. In certain embodiments of the application, the topicalformulation comprises a carrier system. Pharmaceutically effectivecarriers include, but are not limited to, solvents (e.g., alcohols, polyalcohols, water), creams, lotions, ointments, oils, plasters, liposomes,powders, emulsions, microemulsions, and buffered solutions (e.g.,hypotonic or buffered saline) or any other carrier known in the art fortopically administering pharmaceuticals. A more complete listing ofart-known carriers is provided by reference texts that are standard inthe art, for example, Remington's Pharmaceutical Sciences, 16th Edition,1980 and 17th Edition, 1985, both published by Mack Publishing Company,Easton, Pa., the disclosures of which are incorporated herein byreference in their entireties. In certain other embodiments, the topicalformulations of the application may comprise excipients. Anypharmaceutically acceptable excipient known in the art may be used toprepare the inventive pharmaceutically acceptable topical formulations.Examples of excipients that can be included in the topical formulationsof the application include, but are not limited to, preservatives,antioxidants, moisturizers, emollients, buffering agents, solubilizingagents, other penetration agents, skin protectants, surfactants, andpropellants, and/or additional therapeutic agents used in combination tothe inventive compound. Suitable preservatives include, but are notlimited to, alcohols, quaternary amines, organic acids, parabens, andphenols. Suitable antioxidants include, but are not limited to, ascorbicacid and its esters, sodium bisulfite, butylated hydroxytoluene,butylated hydroxyanisole, tocopherols, and chelating agents like EDTAand citric acid. Suitable moisturizers include, but are not limited to,glycerine, sorbitol, polyethylene glycols, urea, and propylene glycol.Suitable buffering agents for use with the application include, but arenot limited to, citric, hydrochloric, and lactic acid buffers. Suitablesolubilizing agents include, but are not limited to, quaternary ammoniumchlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.Suitable skin protectants that can be used in the topical formulationsof the application include, but are not limited to, vitamin E oil,allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

In certain embodiments, the pharmaceutically acceptable topicalformulations of the application comprise at least a compound of theapplication and a penetration enhancing agent. The choice of topicalformulation will depend or several factors, including the condition tobe treated, the physicochemical characteristics of the inventivecompound and other excipients present, their stability in theformulation, available manufacturing equipment, and costs constraints.As used herein the term “penetration enhancing agent” means an agentcapable of transporting a pharmacologically active compound through thestratum corneum and into the epidermis or dermis, preferably, withlittle or no systemic absorption. A wide variety of compounds have beenevaluated as to their effectiveness in enhancing the rate of penetrationof drugs through the skin. See, for example, Percutaneous PenetrationEnhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., BocaRaton, Fla. (1995), which surveys the use and testing of various skinpenetration enhancers, and Buyuktimkin et al., Chemical Means ofTransdermal Drug Permeation Enhancement in Transdermal and Topical DrugDelivery Systems, Gosh T. K., Pfister W. R., Yum S. I. (Eds.),Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplaryembodiments, penetration agents for use with the application include,but are not limited to, triglycerides (e.g., soybean oil), aloecompositions (e.g., aloe-vera gel), ethyl alcohol, isopropyl alcohol,octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400,propylene glycol, N-decylmethylsulfoxide, fatty acid esters (e.g.,isopropyl myristate, methyl laurate, glycerol monooleate, and propyleneglycol monooleate), and N-methylpyrrolidone.

In certain embodiments, the compositions may be in the form ofointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants or patches. In certain exemplary embodiments, formulations ofthe compositions according to the application are creams, which mayfurther contain saturated or unsaturated fatty acids such as stearicacid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleylalcohols, and stearic acid being particularly preferred. Creams of theapplication may also contain a non-ionic surfactant, for example,polyoxy-40-stearate. In certain embodiments, the active component isadmixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this application. Additionally, the presentapplication contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are made by dissolving or dispensing thecompound in the proper medium. As discussed above, penetration enhancingagents can also be used to increase the flux of the compound across theskin. The rate can be controlled by either providing a rate controllingmembrane or by dispersing the compound in a polymer matrix or gel.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present application can be formulated and employedin combination therapies, that is, the compounds and pharmaceuticalcompositions can be formulated with or administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It willalso be appreciated that the therapies employed may achieve a desiredeffect for the same disorder (for example, an inventive compound may beadministered concurrently with another immunomodulatory agent oranticancer agent, or they may achieve different effects (e.g., controlof any adverse effects).

For example, other therapies or anticancer agents that may be used incombination with the compounds of the present application includesurgery, radiotherapy, endocrine therapy, biologic response modifiers(interferons, interleukins, and tumor necrosis factor (TNF) to name afew), hyperthermia and cryotherapy, agents to attenuate any adverseeffects (e.g., antiemetics), and other approved chemotherapeutic drugs,including, but not limited to, alkylating drugs (mechlorethamine,chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites(Methotrexate), purine antagonists and pyrimidine antagonists(6-Mercaptopurine, 5-Fluorouracil, Cytarabile, Gemcitabine), spindlepoisons (Vinblastine, Vincristine, Vinorelbine, Paclitaxel),podophyllotoxins (Etoposide, Irinotecan, Topotecan), antibiotics(Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (Carmustine,Lomustine), inorganic ions (Cisplatin, Carboplatin), enzymes(Asparaginase), and hormones (Tamoxifen, Leuprolide, Flutamide, andMegestrol), to name a few. For a more comprehensive discussion ofupdated cancer therapies see, The Merck Manual, Seventeenth Ed. 1999,the entire contents of which are hereby incorporated by reference. Seealso the National Cancer Institute (CNI) website (www.nci.nih.gov) andthe Food and Drug Administration (FDA) website for a list of the FDAapproved oncology drugs (www.fda.gov/cder/cancer/druglistframe).

In certain embodiments, the pharmaceutical compositions of the presentapplication further comprise one or more additional therapeuticallyactive ingredients (e.g., chemotherapeutic and/or palliative). Forpurposes of the application, the term “palliative” refers to treatmentthat is focused on the relief of symptoms of a disease and/or sideeffects of a therapeutic regimen, but is not curative. For example,palliative treatment encompasses painkillers, antinausea medications andanti-sickness drugs. In addition, chemotherapy, radiotherapy and surgerycan all be used palliatively (that is, to reduce symptoms without goingfor cure; e.g., for shrinking tumors and reducing pressure, bleeding,pain and other symptoms of cancer).

Additionally, the present application provides pharmaceuticallyacceptable derivatives of the inventive compounds, and methods oftreating a subject using these compounds, pharmaceutical compositionsthereof, or either of these in combination with one or more additionaltherapeutic agents.

It will also be appreciated that certain of the compounds of presentapplication can exist in free form for treatment, or where appropriate,as a pharmaceutically acceptable derivative thereof. According to thepresent application, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or a prodrug or other adduct or derivative of a compoundof this application which upon administration to a patient in need iscapable of providing, directly or indirectly, a compound as otherwisedescribed herein, or a metabolite or residue thereof.

Methods of Treatment

In general, methods of using the compounds of the present applicationcomprise administering to a subject in need thereof a therapeuticallyeffective amount of a compound of the present application. The compoundsof the application are generally inducers of target protein degradation.

In certain embodiments, compounds of the application are useful in thetreatment of proliferative diseases (e.g., cancer, benign neoplasms,inflammatory disease, and autoimmune diseases). In certain embodiments,according to the methods of treatment of the present application, levelsof cell proteins of interest, e.g., pathogenic and oncogenic proteinsare modulated, or their growth is inhibited by contacting said cellswith an inventive compound or composition, as described herein. In otherembodiments, the compounds are useful in treating cancer.

Thus, in another aspect of the application, methods for the treatment ofcancer are provided comprising administering a therapeutically effectiveamount of an inventive compound, as described herein, to a subject inneed thereof. In certain embodiments, a method for the treatment ofcancer is provided comprising administering a therapeutically effectiveamount of an inventive compound, or a pharmaceutical compositioncomprising an inventive compound to a subject in need thereof, in suchamounts and for such time as is necessary to achieve the desired result.Preferably, the compounds of present application are administered orallyor intravenously. In certain embodiments of the present application a“therapeutically effective amount” of the inventive compound orpharmaceutical composition is that amount effective for killing orinhibiting the growth of tumor cells. The compounds and compositions,according to the method of the present application, may be administeredusing any amount and any route of administration effective for killingor inhibiting the growth of tumor cells. Thus, the expression “amounteffective to kill or inhibit the growth of tumor cells,” as used herein,refers to a sufficient amount of agent to kill or inhibit the growth oftumor cells. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the infection, the particular anticancer agent,its mode of administration, and the like. In certain embodiments of thepresent application a “therapeutically effective amount” of theinventive compound or pharmaceutical composition is that amounteffective for reducing the levels of target proteins. In certainembodiments of the present application a “therapeutically effectiveamount” of the compound or pharmaceutical composition is that amounteffective to kill or inhibit the growth of skin cells.

In certain embodiments, the method involves the administration of atherapeutically effective amount of the compound or a pharmaceuticallyacceptable derivative thereof to a subject (including, but not limitedto a human or animal) in need of it. In certain embodiments, thebifunctional compounds as useful for the treatment of cancer (including,but not limited to, glioblastoma, retinoblastoma, breast cancer,cervical cancer, colon and rectal cancer, leukemia, lymphoma, lungcancer (including, but not limited to small cell lung cancer), melanomaand/or skin cancer, multiple myeloma, non-Hodgkin's lymphoma, ovariancancer, pancreatic cancer, prostate cancer and gastric cancer, bladdercancer, uterine cancer, kidney cancer, testicular cancer, stomachcancer, brain cancer, liver cancer, or esophageal cancer.

In certain embodiments, the inventive anticancer agents are useful inthe treatment of cancers and other proliferative disorders, including,but not limited to breast cancer, cervical cancer, colon and rectalcancer, leukemia, lung cancer, melanoma, multiple myeloma, non-Hodgkin'slymphoma, ovarian cancer, pancreatic cancer, prostate cancer, andgastric cancer. In certain embodiments, the inventive anticancer agentsare active against solid tumors.

In certain embodiments, the inventive compounds also find use in theprevention of restenosis of blood vessels subject to traumas such asangioplasty and stenting. For example, it is contemplated that thecompounds of the application will be useful as a coating for implantedmedical devices, such as tubings, shunts, catheters, artificialimplants, pins, electrical implants such as pacemakers, and especiallyfor arterial or venous stents, including balloon-expandable stents. Incertain embodiments inventive compounds may be bound to an implantablemedical device, or alternatively, may be passively adsorbed to thesurface of the implantable device. In certain other embodiments, theinventive compounds may be formulated to be contained within, or,adapted to release by a surgical or medical device or implant, such as,for example, stents, sutures, indwelling catheters, prosthesis, and thelike. For example, drugs having antiproliferative and anti-inflammatoryactivities have been evaluated as stent coatings, and have shown promisein preventing retenosis (See, for example, Presbitero P. et al., “Drugeluting stents do they make the difference?”, Minerva Cardioangiol,2002, 50(5):431-442; Ruygrok P. N. et al., “Rapamycin in cardiovascularmedicine”, Intern. Med. J., 2003, 33(3):103-109; and Marx S. O. et al.,“Bench to bedside: the development of rapamycin and its application tostent restenosis”, Circulation, 2001, 104(8):852-855, each of thesereferences is incorporated herein by reference in its entirety).Accordingly, without wishing to be bound to any particular theory,Applicant proposes that inventive compounds having antiproliferativeeffects can be used as stent coatings and/or in stent drug deliverydevices, inter alia for the prevention of restenosis or reduction ofrestenosis rate. Suitable coatings and the general preparation of coatedimplantable devices are described in U.S. Pat. Nos. 6,099,562;5,886,026; and 5,304,121. The coatings are typically biocompatiblepolymeric materials such as a hydrogel polymer, polymethyldisiloxane,polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinylacetate, and mixtures thereof. The coatings may optionally be furthercovered by a suitable topcoat of fluorosilicone, polysaccarides,polyethylene glycol, phospholipids or combinations thereof to impartcontrolled release characteristics in the composition. A variety ofcompositions and methods related to stent coating and/or local stentdrug delivery for preventing restenosis are known in the art (see, forexample, U.S. Pat. Nos. 6,517,889; 6,273,913; 6,258,121; 6,251,136;6,248,127; 6,231,600; 6,203,551; 6,153,252; 6,071,305; 5,891,507;5,837,313 and published U.S. patent application No.: US2001/0027340,each of which is incorporated herein by reference in its entirety). Forexample, stents may be coated with polymer-drug conjugates by dippingthe stent in polymer-drug solution or spraying the stent with such asolution. In certain embodiment, suitable materials for the implantabledevice include biocompatible and nontoxic materials, and may be chosenfrom the metals such as nickel-titanium alloys, steel, or biocompatiblepolymers, hydrogels, polyurethanes, polyethylenes, ethylenevinyl acetatecopolymers, etc. In certain embodiments, the inventive compound iscoated onto a stent for insertion into an artery or vein followingballoon angioplasty.

The compounds of this application or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating implantable medical devices, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentapplication, in another aspect, includes a composition for coating animplantable device comprising a compound of the present application asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present application includes an implantable device coatedwith a composition comprising a compound of the present application asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device.

Additionally, the present application provides pharmaceuticallyacceptable derivatives of the inventive compounds, and methods oftreating a subject using these compounds, pharmaceutical compositionsthereof, or either of these in combination with one or more additionaltherapeutic agents.

Another aspect of the application relates to a method of treating orlessening the severity of a disease or condition associated with aproliferation disorder in a patient, said method comprising a step ofadministering to said patient, a compound of Formula I or a compositioncomprising said compound.

It will be appreciated that the compounds and compositions, according tothe method of the present application, may be administered using anyamount and any route of administration effective for the treatment ofcancer and/or disorders associated with cell hyperproliferation. Forexample, when using the inventive compounds for the treatment of cancer,the expression “effective amount” as used herein, refers to a sufficientamount of agent to inhibit cell proliferation, or refers to a sufficientamount to reduce the effects of cancer. The exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the diseases, the particularanticancer agent, its mode of administration, and the like.

The compounds of the application are preferably formulated in dosageunit form for ease of administration and uniformity of dosage. Theexpression “dosage unit form” as used herein refers to a physicallydiscrete unit of therapeutic agent appropriate for the patient to betreated. It will be understood, however, that the total daily usage ofthe compounds and compositions of the present application will bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically effective dose level for anyparticular patient or organism will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts (see,for example, Goodman and Gilman's, “The Pharmacological Basis ofTherapeutics”, Tenth Edition, A. Gilman, J. Hardman and L. Limbird,eds., McGraw-Hill Press, 155-173, 2001, which is incorporated herein byreference in its entirety).

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable carrier in a desired dosage, the pharmaceutical compositionsof this application can be administered to humans and other animalsorally, rectally, parenterally, intracisternally, intravaginally,intraperitoneally, topically (as by powders, ointments, creams ordrops), bucally, as an oral or nasal spray, or the like, depending onthe severity of the infection being treated. In certain embodiments, thecompounds of the application may be administered at dosage levels ofabout 0.001 mg/kg to about 50 mg/kg, from about 0.01 mg/kg to about 25mg/kg, or from about 0.1 mg/kg to about 10 mg/kg of subject body weightper day, one or more times a day, to obtain the desired therapeuticeffect. It will also be appreciated that dosages smaller than 0.001mg/kg or greater than 50 mg/kg (for example 50-100 mg/kg) can beadministered to a subject. In certain embodiments, compounds areadministered orally or parenterally.

The present application provides methods for the treatment of a cellproliferative disorder in a subject in need thereof by administering toa subject in need of such treatment, a therapeutically effective amountof a compound of the present application, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof. Thecell proliferative disorder can be cancer or a precancerous condition.The present application further provides the use of a compound of thepresent application, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, for the preparation of amedicament useful for the treatment of a cell proliferative disorder.

The present application also provides methods of protecting against acell proliferative disorder in a subject in need thereof byadministering a therapeutically effective amount of compound of thepresent application, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, to a subject in need of suchtreatment. The cell proliferative disorder can be cancer or aprecancerous condition. The present application also provides the use ofcompound of the present application, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, for thepreparation of a medicament useful for the prevention of a cellproliferative disorder.

As used herein, a “subject in need thereof” is a subject having a cellproliferative disorder, or a subject having an increased risk ofdeveloping a cell proliferative disorder relative to the population atlarge. A subject in need thereof can have a precancerous condition.Preferably, a subject in need thereof has cancer. A “subject” includes amammal. The mammal can be e.g., any mammal, e.g., a human, primate,bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or apig. Preferably, the mammal is a human.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders ofthe application encompass a variety of conditions wherein cell divisionis deregulated. Exemplary cell proliferative disorder include, but arenot limited to, neoplasms, benign tumors, malignant tumors,pre-cancerous conditions, in situ tumors, encapsulated tumors,metastatic tumors, liquid tumors, solid tumors, immunological tumors,hematological tumors, cancers, carcinomas, leukemias, lymphomas,sarcomas, and rapidly dividing cells. The term “rapidly dividing cell”as used herein is defined as any cell that divides at a rate thatexceeds or is greater than what is expected or observed amongneighboring or juxtaposed cells within the same tissue. A cellproliferative disorder includes a precancer or a precancerous condition.A cell proliferative disorder includes cancer. Preferably, the methodsprovided herein are used to treat or alleviate a symptom of cancer. Theterm “cancer” includes solid tumors, as well as, hematologic tumorsand/or malignancies. A “precancer cell” or “precancerous cell” is a cellmanifesting a cell proliferative disorder that is a precancer or aprecancerous condition. A “cancer cell” or “cancerous cell” is a cellmanifesting a cell proliferative disorder that is a cancer. Anyreproducible means of measurement may be used to identify cancer cellsor precancerous cells. Cancer cells or precancerous cells can beidentified by histological typing or grading of a tissue sample (e.g., abiopsy sample). Cancer cells or precancerous cells can be identifiedthrough the use of appropriate molecular markers.

Exemplary non-cancerous conditions or disorders include, but are notlimited to, rheumatoid arthritis; inflammation; autoimmune disease;lymphoproliferative conditions; acromegaly; rheumatoid spondylitis;osteoarthritis; gout, other arthritic conditions; sepsis; septic shock;endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma;adult respiratory distress syndrome; chronic obstructive pulmonarydisease; chronic pulmonary inflammation; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreaticfibrosis; hepatic fibrosis; acute and chronic renal disease; irritablebowel syndrome; pyresis; restenosis; cerebral malaria; stroke andischemic injury; neural trauma; Alzheimer's disease; Huntington'sdisease; Parkinson's disease; acute and chronic pain; allergic rhinitis;allergic conjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; and diabetes mellitus.

Exemplary cancers include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, appendix cancer, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, basal cellcarcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bileduct cancer, intrahepatic bile duct cancer, bladder cancer, uringarybladder cancer, bone and joint cancer, osteosarcoma and malignantfibrous histiocytoma, brain cancer, brain tumor, brain stem glioma,cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodeimaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer,laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia,chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cellleukemia, lip and oral cavity cancer, liver cancer, lung cancer,non-small cell lung cancer, small cell lung cancer, AIDS-relatedlymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma,Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular(eye) melanoma, merkel cell carcinoma, mesothelioma malignant,mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer ofthe tongue, multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterinecancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer(melanoma), merkel cell skin carcinoma, small intestine cancer, softtissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, testicular cancer,throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter and otherurinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, and Wilm's Tumor.

A “cell proliferative disorder of the hematologic system” is a cellproliferative disorder involving cells of the hematologic system. A cellproliferative disorder of the hematologic system can include lymphoma,leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benignmonoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoidpapulosis, polycythemia vera, chronic myelocytic leukemia, agnogenicmyeloid metaplasia, and essential thrombocythemia. A cell proliferativedisorder of the hematologic system can include hyperplasia, dysplasia,and metaplasia of cells of the hematologic system. Preferably,compositions of the present application may be used to treat a cancerselected from the group consisting of a hematologic cancer of thepresent application or a hematologic cell proliferative disorder of thepresent application. A hematologic cancer of the present application caninclude multiple myeloma, lymphoma (including Hodgkin's lymphoma,non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas oflymphocytic and cutaneous origin), leukemia (including childhoodleukemia, hairy-cell leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, chronic lymphocytic leukemia, chronic myelocyticleukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloidneoplasms and mast cell neoplasms.

A “cell proliferative disorder of the lung” is a cell proliferativedisorder involving cells of the lung. Cell proliferative disorders ofthe lung can include all forms of cell proliferative disorders affectinglung cells. Cell proliferative disorders of the lung can include lungcancer, a precancer or precancerous condition of the lung, benigngrowths or lesions of the lung, and malignant growths or lesions of thelung, and metastatic lesions in tissue and organs in the body other thanthe lung. Preferably, compositions of the present application may beused to treat lung cancer or cell proliferative disorders of the lung.Lung cancer can include all forms of cancer of the lung. Lung cancer caninclude malignant lung neoplasms, carcinoma in situ, typical carcinoidtumors, and atypical carcinoid tumors. Lung cancer can include smallcell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”),squamous cell carcinoma, adenocarcinoma, small cell carcinoma, largecell carcinoma, adenosquamous cell carcinoma, and mesothelioma. Lungcancer can include “scar carcinoma”, bronchioalveolar carcinoma, giantcell carcinoma, spindle cell carcinoma, and large cell neuroendocrinecarcinoma. Lung cancer can include lung neoplasms having histologic andultrastructual heterogeneity (e.g., mixed cell types).

Cell proliferative disorders of the lung can include all forms of cellproliferative disorders affecting lung cells. Cell proliferativedisorders of the lung can include lung cancer, precancerous conditionsof the lung. Cell proliferative disorders of the lung can includehyperplasia, metaplasia, and dysplasia of the lung. Cell proliferativedisorders of the lung can include asbestos-induced hyperplasia, squamousmetaplasia, and benign reactive mesothelial metaplasia. Cellproliferative disorders of the lung can include replacement of columnarepithelium with stratified squamous epithelium, and mucosal dysplasia.Individuals exposed to inhaled injurious environmental agents such ascigarette smoke and asbestos may be at increased risk for developingcell proliferative disorders of the lung. Prior lung diseases that maypredispose individuals to development of cell proliferative disorders ofthe lung can include chronic interstitial lung disease, necrotizingpulmonary disease, scleroderma, rheumatoid disease, sarcoidosis,interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathicpulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, andHodgkin's disease.

A “cell proliferative disorder of the colon” is a cell proliferativedisorder involving cells of the colon. Preferably, the cellproliferative disorder of the colon is colon cancer. Preferably,compositions of the present application may be used to treat coloncancer or cell proliferative disorders of the colon. Colon cancer caninclude all forms of cancer of the colon. Colon cancer can includesporadic and hereditary colon cancers. Colon cancer can includemalignant colon neoplasms, carcinoma in situ, typical carcinoid tumors,and atypical carcinoid tumors. Colon cancer can include adenocarcinoma,squamous cell carcinoma, and adenosquamous cell carcinoma. Colon cancercan be associated with a hereditary syndrome selected from the groupconsisting of hereditary nonpolyposis colorectal cancer, familialadenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome,Turcot's syndrome and juvenile polyposis. Colon cancer can be caused bya hereditary syndrome selected from the group consisting of hereditarynonpolyposis colorectal cancer, familial adenomatous polyposis,Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome andjuvenile polyposis.

Cell proliferative disorders of the colon can include all forms of cellproliferative disorders affecting colon cells. Cell proliferativedisorders of the colon can include colon cancer, precancerous conditionsof the colon, adenomatous polyps of the colon and metachronous lesionsof the colon. A cell proliferative disorder of the colon can includeadenoma. Cell proliferative disorders of the colon can be characterizedby hyperplasia, metaplasia, and dysplasia of the colon. Prior colondiseases that may predispose individuals to development of cellproliferative disorders of the colon can include prior colon cancer.Current disease that may predispose individuals to development of cellproliferative disorders of the colon can include Crohn's disease andulcerative colitis. A cell proliferative disorder of the colon can beassociated with a mutation in a gene selected from the group consistingof p53, ras, FAP and DCC. An individual can have an elevated risk ofdeveloping a cell proliferative disorder of the colon due to thepresence of a mutation in a gene selected from the group consisting ofp53, ras, FAP and DCC.

A “cell proliferative disorder of the pancreas” is a cell proliferativedisorder involving cells of the pancreas. Cell proliferative disordersof the pancreas can include all forms of cell proliferative disordersaffecting pancreatic cells. Cell proliferative disorders of the pancreascan include pancreas cancer, a precancer or precancerous condition ofthe pancreas, hyperplasia of the pancreas, and dysaplasia of thepancreas, benign growths or lesions of the pancreas, and malignantgrowths or lesions of the pancreas, and metastatic lesions in tissue andorgans in the body other than the pancreas. Pancreatic cancer includesall forms of cancer of the pancreas. Pancreatic cancer can includeductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cellcarcinoma, mucinous adenocarcinoma, osteoclast-like giant cellcarcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassifiedlarge cell carcinoma, small cell carcinoma, pancreatoblastoma, papillaryneoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serouscystadenoma. Pancreatic cancer can also include pancreatic neoplasmshaving histologic and ultrastructual heterogeneity (e.g., mixed celltypes).

A “cell proliferative disorder of the prostate” is a cell proliferativedisorder involving cells of the prostate. Cell proliferative disordersof the prostate can include all forms of cell proliferative disordersaffecting prostate cells. Cell proliferative disorders of the prostatecan include prostate cancer, a precancer or precancerous condition ofthe prostate, benign growths or lesions of the prostate, and malignantgrowths or lesions of the prostate, and metastatic lesions in tissue andorgans in the body other than the prostate. Cell proliferative disordersof the prostate can include hyperplasia, metaplasia, and dysplasia ofthe prostate.

A “cell proliferative disorder of the skin” is a cell proliferativedisorder involving cells of the skin. Cell proliferative disorders ofthe skin can include all forms of cell proliferative disorders affectingskin cells. Cell proliferative disorders of the skin can include aprecancer or precancerous condition of the skin, benign growths orlesions of the skin, melanoma, malignant melanoma and other malignantgrowths or lesions of the skin, and metastatic lesions in tissue andorgans in the body other than the skin. Cell proliferative disorders ofthe skin can include hyperplasia, metaplasia, and dysplasia of the skin.

A “cell proliferative disorder of the ovary” is a cell proliferativedisorder involving cells of the ovary. Cell proliferative disorders ofthe ovary can include all forms of cell proliferative disordersaffecting cells of the ovary. Cell proliferative disorders of the ovarycan include a precancer or precancerous condition of the ovary, benigngrowths or lesions of the ovary, ovarian cancer, malignant growths orlesions of the ovary, and metastatic lesions in tissue and organs in thebody other than the ovary. Cell proliferative disorders of the skin caninclude hyperplasia, metaplasia, and dysplasia of cells of the ovary.

A “cell proliferative disorder of the breast” is a cell proliferativedisorder involving cells of the breast. Cell proliferative disorders ofthe breast can include all forms of cell proliferative disordersaffecting breast cells. Cell proliferative disorders of the breast caninclude breast cancer, a precancer or precancerous condition of thebreast, benign growths or lesions of the breast, and malignant growthsor lesions of the breast, and metastatic lesions in tissue and organs inthe body other than the breast. Cell proliferative disorders of thebreast can include hyperplasia, metaplasia, and dysplasia of the breast.

A cancer that is to be treated can be staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of TX, T₁, T₁mic, T₁a, T₁b, T₁c, T₂,T₃, T₄, T₄a, T₄b, T₄c, or T₄d; and where the regional lymph nodes (N)have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, orN3c; and where distant metastasis (M) can be assigned a stage of MX, M0,or M1. A cancer that is to be treated can be staged according to anAmerican Joint Committee on Cancer (AJCC) classification as Stage I,Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. Acancer that is to be treated can be assigned a grade according to anAJCC classification as Grade GX (e.g., grade cannot be assessed), Grade1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can bestaged according to an AJCC pathologic classification (pN) of pNX, pN0,PN0 (I−), PN0 (I+), PN0 (mol−), PN0 (mol+), PN1, PN1(mi), PN1a, PN1b,PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

A cancer that is to be treated can include a tumor that has beendetermined to be less than or equal to about 2 centimeters in diameter.A cancer that is to be treated can include a tumor that has beendetermined to be from about 2 to about 5 centimeters in diameter. Acancer that is to be treated can include a tumor that has beendetermined to be greater than or equal to about 3 centimeters indiameter. A cancer that is to be treated can include a tumor that hasbeen determined to be greater than 5 centimeters in diameter. A cancerthat is to be treated can be classified by microscopic appearance aswell differentiated, moderately differentiated, poorly differentiated,or undifferentiated. A cancer that is to be treated can be classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Acancer that is to be treated can be classified by microscopic appearanceas being associated with areas of necrosis (e.g., areas of dying ordegenerating cells). A cancer that is to be treated can be classified ashaving an abnormal karyotype, having an abnormal number of chromosomes,or having one or more chromosomes that are abnormal in appearance. Acancer that is to be treated can be classified as being aneuploid,triploid, tetraploid, or as having an altered ploidy. A cancer that isto be treated can be classified as having a chromosomal translocation,or a deletion or duplication of an entire chromosome, or a region ofdeletion, duplication or amplification of a portion of a chromosome.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cellsin the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder”. A normal cell lacks unregulatedor abnormal growth, or both, that can lead to the development of anunwanted condition or disease. Preferably, a normal cell possessesnormally functioning cell cycle checkpoint control mechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter is in direct contact with acell, or is close enough to induce a desired biological effect in acell.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentapplication, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, to alleviate the symptoms or complicationsof a disease, condition or disorder, or to eliminate the disease,condition or disorder.

A compound of the present application, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can also beused to prevent a disease, condition or disorder. As used herein,“preventing” or “prevent” describes reducing or eliminating the onset ofthe symptoms or complications of the disease, condition or disorder.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. In a preferred embodiment, the administration ofpharmaceutical compositions of the application leads to the eliminationof a sign or symptom, however, elimination is not required. Effectivedosages are expected to decrease the severity of a sign or symptom. Forinstance, a sign or symptom of a disorder such as cancer, which canoccur in multiple locations, is alleviated if the severity of the canceris decreased within at least one of multiple locations.

The compounds described herein (e.g., the bifunctional compounds), onceproduced, can be characterized using a variety of assays known to thoseskilled in the art to determine whether the compounds have the desiredbiological activity. For example, the molecules can be characterized byconventional assays, including but not limited to those assays describedbelow (e.g., treating cells of interest, such as MV4-11 cells, humancell line MM1S, or a human cell line MM1S that is deficient in cereblon,with a test compound and then performing immunoblotting against theindicated proteins such as BRD2, BRD3, and BRD4, or treating certaincells of interest with a test compound and then measuring BRD4transcript levels via qRT-PCR), to determine whether they have apredicted activity, binding activity and/or binding specificity.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual(3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the application

Definitions

Certain compounds of the present application, and definitions ofspecific functional groups are also described in more detail below.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisapplication, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. For purposes of this application, heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalencies of the heteroatoms.

The term “Linker”, “linker”, “Linker group” or “linker group” as usedherein, refers to a chemical moiety utilized to attach one part of acompound of interest to another compound of interest. These bindingmoieties of the present application are linked to the ubiquitin ligasebinding moiety preferably through a Linker in order to present a targetprotein (to which the protein target moiety is bound) in proximity tothe ubiquitin ligase for ubiquitination and degradation. ExemplaryLinkers are described herein.

The term “compound” or “chemical compound” as used herein can includeorganometallic compounds, organic compounds, metals, transitional metalcomplexes, and small molecules. In certain preferred embodiments,polynucleotides are excluded from the definition of compounds. In otherpreferred embodiments, polynucleotides and peptides are excluded fromthe definition of compounds. In a particularly preferred embodiment, theterm compounds refers to small molecules (e.g., preferably, non-peptidicand non-oligomeric) and excludes peptides, polynucleotides, transitionmetal complexes, metals, and organometallic compounds.

As used herein, the term “small molecule” refers to a non-peptidic,non-oligomeric organic compound either synthesized in the laboratory orfound in nature. Small molecules, as used herein, can refer to compoundsthat are “natural product-like”, however, the term “small molecule” isnot limited to “natural product-like” compounds. Rather, a smallmolecule is typically characterized in that it contains severalcarbon-carbon bonds, and has a molecular weight of less than 2000 g/mol,preferably less than 1500 g/mol, although this characterization is notintended to be limiting for the purposes of the present application. Incertain other preferred embodiments, synthetic small molecules areutilized.

The term “independently” is used herein to indicate that the variable,such as atom or functional group, which is independently applied, variesindependently from application to application. For example, where morethan one substituent or atom (carbon or heteroatom, such as oxygen (O),sulfur (S), or nitrogen (N)) occurs, each substituent or atom isindependent of another substituent or atom and such substituents or atomcan also alternate.

In chemistry, a “derivative” is a compound that is derived from asimilar compound by some chemical or physical process. It is also usedto mean that a compound can arise from another compound, if one atom isreplaced with another atom or group of atoms. A term “structuralanalogue” can be also used for this meaning.

The term “structural analogue” or term “analogue” has always been usedto describe structural and functional similarity. Extended to drugs,this definition implies that the analogue of an existing drug moleculeshares structural and pharmacological similarities with the originalcompound. Formally, this definition allows the establishment of threecategories of drug analogues: analogues possessing chemical andpharmacological similarities (direct analogues); analogues possessingstructural similarities only (structural analogues); and chemicallydifferent compounds displaying similar pharmacological properties(functional analogues). For example, lenalidomide and pomalidomide areamong thalidomide analogs, and are believed to act in a similar fashion.

The term “E3 Ubiquitin Ligase” or “Ubiquitin Ligase” (UL) is used hereinto describe a target enzyme(s) binding site of ubiquitin ligase moietiesin the bifunctional compounds according to the present application. E3UL is a protein that in combination with an E2 ubiquitin-conjugatingenzyme causes the attachment of ubiquitin to a lysine on a targetprotein; the E3 ubiquitin ligase targets specific protein substrates fordegradation by the proteasome. Thus, E3 ubiquitin ligase alone or incomplex with an E2 ubiquitin conjugating enzyme is responsible for thetransfer of ubiquitin to targeted proteins. In general, the ubiquitinligase is involved in polyubiquitination such that a second ubiquitin isattached to the first, a third is attached to the second, and so forth.Polyubiquitination marks proteins for degradation by the proteasome.However, there are some ubiquitination events that are limited tomonoubiquitination, in which only a single ubiquitin is added by theubiquitin ligase to a substrate molecule. Mono-ubiquitinated proteinsare not targeted to the proteasome for degradation, but may instead bealtered in their cellular location or function, for example, via bindingother proteins that have domains capable of binding ubiquitin. Furthercomplicating matters, different lysines on ubiquitin can be targeted byan E3 to make chains. The most common lysine is Lys48 on the ubiquitinchain. This is the lysine used to make polyubiquitin, which isrecognized by the proteasome.

The term “protein target moiety” or “target protein ligand” is usedherein to describe a small molecule, which is capable of binding to orbinds to a target protein or other protein or polypeptide of interestand places/presents that protein or polypeptide in proximity to anubiquitin ligase such that degradation of the protein or polypeptide byubiquitin ligase may occur. Any protein, which can bind to a proteintarget moiety and acted on or degraded by an ubiquitin ligase is atarget protein according to the present application. In general, targetproteins may include, for example, structural proteins, receptors,enzymes, cell surface proteins, proteins pertinent to the integratedfunction of a cell, including proteins involved in catalytic activity,aromatase activity, motor activity, helicase activity, metabolicprocesses (anabolism and catrabolism), antioxidant activity,proteolysis, biosynthesis, proteins with kinase activity, oxidoreductaseactivity, transferase activity, hydrolase activity, lyase activity,isomerase activity, ligase activity, enzyme regulator activity, signaltransducer activity, structural molecule activity, binding activity(protein, lipid carbohydrate), receptor activity, cell motility,membrane fusion, cell communication, regulation of biological processes,development, cell differentiation, response to stimulus, behavioralproteins, cell adhesion proteins, proteins involved in cell death,proteins involved in transport (including protein transporter activity,nuclear transport, ion transporter activity, channel transporteractivity, carrier activity, permease activity, secretion activity,electron transporter activity, pathogenesis, chaperone regulatoractivity, nucleic acid binding activity, transcription regulatoractivity, extracellular organization and biogenesis activity,translation regulator activity. Proteins of interest can includeproteins from eurkaryotes and prokaryotes including humans as targetsfor drug therapy, other animals, including domesticated animals,microbials for the determination of targets for antibiotics and otherantimicrobials and plants, and even viruses, among numerous others.Non-limiting examples of small molecule target protein binding moietiesinclude Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compoundstargeting Human BET Bromodomain-containing proteins, HDAC inhibitors,human lysine methyltransferase inhibitors, angiogenesis inhibitors,immunosuppressive compounds, and compounds targeting the arylhydrocarbon receptor (AHR), among numerous others. The compositionsdescribed below exemplify some of the members of these small moleculetarget protein.

As used herein, the term “BRD4” or “Brd4” relates toBromodomain-containing protein 4 is a protein that in humans is encodedby the BRD4 gene. BDR4 is a member of the BET (bromodomain and extraterminal domain) family, along with BRD2, BRD3, and BRDT. BRD4, similarto its BET family members, contains two bromodomains that recognizeacetylated lysine residues. An increase in Brd4 expression led toincreased P-TEFb-dependent phosphorylation of RNA polymerase II (RNAPII)CTD and stimulation of transcription from promoters in vivo. Conversely,a reduction in Brd4 expression by siRNA reduced CTD phosphorylation andtranscription, revealing that Brd4 is a positive regulatory component ofP-TEFb. In chromatin immunoprecipitation (ChIP) assays, the recruitmentof P-TEFb to a promoter was dependent on Brd4 and was enhanced by anincrease in chromatin acetylation. Together, P-TEFb alternatelyinteracts with Brd4 and the inhibitory subunit to maintain functionalequilibrium in the cell.

BRD4 is an exemplary, non-enzymatic protein target. BRD4 is atranscriptional co-activator involved in dynamic transcriptionalactivation and elongation. BRD4 binds to enhancer and promoter regionsadjacent to target genes, via recognition of side-chain acetylatedlysine on histone proteins and transcription factors (TFs) by twinacetyl-lysine binding modules or bromodomains. Recently, a firstdirect-acting inhibitor of BET bromodomains (JQ1) was developed (P.Filippakopoulos et al., Nature 468, 1067-1073 (2010)), that displacesBRD4 from chromatin leading to impaired signal transduction from masterregulatory TFs to RNA Polymerase II (B. Chapuy et al., Cancer Cell 24,777-790 (2013); J. E. Delmore et al., Cell 146, 904-917 (2011); J. Lovenet al., Cell 153, 320-334 (2013).). Molecular recognition of the BRD4bromodomains by JQ1 is stereo-specific, and only the (+)-JQ1 enantiomer(JQ1S; from here forward JQ1) is active; the (−)-JQ1 enantiomer (JQ1R)is inactive. Silencing of BRD4 expression by RNA interference in murineand human models of MM and acute myeloid leukemia (AML) elicited rapidtranscriptional downregulation of the MYC oncogene and a potentanti-proliferative response (J. E. Delmore et al., Cell 146, 904-917(2011); J. Zuber et al., Nature 478, 524-528 (2011)). These and otherstudies in cancer, inflammation (E. Nicodeme et al., Nature 468,1119-1123 (2010)) and heart disease (P. Anand et al., Cell 154, 569-582(2013); J. D. Brown et al., Mol. Cell 56, 219-231 (2014)), establish adesirable mechanistic and translational purpose to target BRD4 forselective degradation.

As used herein, the term “FKBP” relates to a family of proteins thathave prolyl isomerase activity and are related to the cyclophilins infunction, though not in amino acid sequence (Siekierka et al. Nature 341(6244): 755-7 (1989)). FKBPs have been identified in many eukaryotesfrom yeast to humans and function as protein folding chaperones forproteins containing proline residues. Along with cyclophilin, FKBPsbelong to the immunophilin family (Balbach et al. Mechanisms of proteinfolding (2nd ed.). Oxford: Oxford University Press. pp. 212-237 (2000)).Cytosolic signaling protein FKBP12 is notable in humans for binding theimmunosuppressant molecule tacrolimus (originally designated FK506),which is used in treating patients after organ transplant and patientssuffering from autoimmune disorders (Wang et al. Science 265 (5172):674-6 (1994)). Tacrolimus has been found to reduce episodes of organrejection over a related treatment, the drug ciclosporin, which bindscyclophilin (Mayer et al. Transplantation 64 (3): 436-43 (1997)). Boththe FKBP-tacrolimus complex and the ciclosporin-cyclophilin complexinhibit a phosphatase called calcineurin, thus blocking signaltransduction in the T-lymphocytetransduction pathway (Liu et al. Cell 66(4): 807-15 (1991)). This therapeutic role is not related to prolylisomerase activity. AP1497 (Table 1, TL5) is a synthetic pipecolylα-ketoamide designed to be recognized by FKBP12 (Holt et al., J. Am.Chem. Soc. 115, 9925 (1993))

As used herein the term “CREBBP” relates to CREB binding protein. Thisgene is ubiquitously expressed and is involved in the transcriptionalcoactivation of many different transcription factors. First isolated asa nuclear protein that binds to cAMP-response element binding protein(CREB), this gene is now known to play critical roles in embryonicdevelopment, growth control, and homeostasis by coupling chromatinremodeling to transcription factor recognition. Chromosomaltranslocations involving this gene have been associated with acutemyeloid leukemia.

As used herein the term “SMARCA4” relates to transcription activatorBRG1 also known as ATP-dependent helicase SMARCA4 is a protein that inhumans is encoded by the SMARCA4 gene. Mutations in this gene were firstrecognized in human lung cancer cell lines. It has been demonstratedthat BRG1 plays a role in the control of retinoic acid andglucocorticoid-induced cell differentiation in lung cancer and in othertumor types.

As used herein the term “nuclear receptor” relates to a class ofproteins found within cells that are responsible for sensing steroid andthyroid hormones and certain other molecules. In response, thesereceptors work with other proteins to regulate the expression ofspecific genes, thereby controlling the development, homeostasis, andmetabolism of the organism. Since the expression of a large number ofgenes is regulated by nuclear receptors, ligands that activate thesereceptors can have profound effects on the organism.

The representative examples which follow are intended to help illustratethe application, and are not intended to, nor should they be construedto, limit the scope of the application. Indeed, various modifications ofthe application and many further embodiments thereof, in addition tothose shown and described herein, will become apparent to those skilledin the art from the full contents of this document, including theexamples which follow and the references to the scientific and patentliterature cited herein. It should further be appreciated that, unlessotherwise indicated, the entire contents of each of the references citedherein are incorporated herein by reference to help illustrate the stateof the art. The following examples contain important additionalinformation, exemplification and guidance which can be adapted to thepractice of this application in its various embodiments and theequivalents thereof.

These and other aspects of the present application will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the applicationbut are not intended to limit its scope, as defined by the claims.

EXAMPLES

General Description of Synthetic Methods

The various references cited herein provide helpful backgroundinformation on preparing compounds similar to the inventive compoundsdescribed herein or relevant intermediates, as well as information onformulation, uses, and administration of such compounds which may be ofinterest.

Moreover, the practitioner is directed to the specific guidance andexamples provided in this document relating to various exemplarycompounds and intermediates thereof.

According to the present application, any available techniques can beused to make or prepare the inventive compounds or compositionsincluding them. For example, a variety of a variety combinatorialtechniques, parallel synthesis and/or solid phase synthetic methods suchas those discussed in detail below may be used. Alternatively oradditionally, the inventive compounds may be prepared using any of avariety of solution phase synthetic methods known in the art.

The starting materials, intermediates, and compounds of this applicationmay be isolated and purified using conventional techniques, includingfiltration, distillation, crystallization, chromatography, and the like.They may be characterized using conventional methods, including physicalconstants and spectral data.

Synthesis of Exemplary Compounds

Unless otherwise indicated, starting materials are either commerciallyavailable or readily accessible through laboratory synthesis by anyonereasonably familiar with the art. Described generally below, areprocedures and general guidance for the synthesis of compounds asdescribed generally and in subclasses and species herein.

Example 1: Synthesis of dBET1

(1) Synthesis of JQ-Acid

JQ1 (1.0 g, 2.19 mmol, 1 eq) was dissolved in formic acid (11 mL, 0.2 M)at room temperature and stirred for 75 hours. The mixture wasconcentrated under reduced pressure to give a yellow solid (0.99 g,quant yield) that was used without purification. ¹H NMR (400 MHz,Methanol-d₄) δ 7.50-7.36 (m, 4H), 4.59 (t, J=7.1 Hz, 1H), 3.51 (d, J=7.1Hz, 2H), 2.70 (s, 3H), 2.45 (s, 3H), 1.71 (s, 3H). LCMS 401.33 (M+H).

N-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetatewas synthesized according to the previously published procedure (Fischeret al. Nature 2014, 512, 49).

(2) Synthesis of dBET1

JQ-acid (11.3 mg, 0.0281 mmol, 1 eq) andN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate (14.5 mg, 0.0281 mmol, 1 eq) were dissolved in DMF(0.28 mL, 0.1 M) at room temperature. DIPEA (14.7 microliters, 0.0843mmol, 3 eq) and HATU (10.7 mg, 0.0281 mmol, 1 eq) were then added andthe mixture was stirred for 19 hours. The mixture was then purified bypreparative HPLC to give dBET1 as a yellow solid (15.90 mg, 0.0202 mmol,72%). ¹H NMR (400 MHz, Methanol-d₄) δ 7.77 (dd, J=8.3, 7.5 Hz, 1H), 7.49(d, J=7.3 Hz, 1H), 7.47-7.37 (m, 5H), 5.07 (dd, J=12.5, 5.4 Hz, 1H),4.74 (s, 2H), 4.69 (dd, J=8.7, 5.5 Hz, 1H), 3.43-3.32 (m, 3H), 3.29-3.25(m, 2H), 2.87-2.62 (m, 7H), 2.43 (s, 3H), 2.13-2.04 (m, 1H), 1.72-1.58(m, 7H). ¹³C NMR (100 MHz, cd₃od) δ 174.41, 172.33, 171.27, 171.25,169.87, 168.22, 167.76, 166.73, 166.70, 156.26, 138.40, 138.23, 137.44,134.83, 133.92, 133.40, 132.30, 132.28, 131.97, 131.50, 129.87, 121.85,119.31, 118.00, 69.53, 54.90, 50.54, 40.09, 39.83, 38.40, 32.12, 27.74,27.65, 23.61, 14.42, 12.97, 11.57. LCMS 785.44 (M+H).

Example 2: Synthesis of dBET4

A 0.1 M solution ofN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.438 mL, 0.0438 mmol 1.2 eq) was added to(R)-JQ-acid (prepared from (R)-JQ1 in an analogous method to JQ-acid)(14.63 mg, 0.0365 mmol, 1 eq) at room temperature. DIPEA (19.1microliters, 0.1095 mmol, 3 eq) and HATU (15.3 mg, 0.0402 mmol, 1.1 eq)were added and the mixture was stirred for 24 hours, then diluted withMeOH and concentrated under reduced pressure. The crude material waspurified by preparative HPLC to give a yellow solid (20.64 mg, 0.0263mmol, 72%). ¹H NMR (400 MHz, Methanol-d₄) δ 7.79 (dd, J=8.4, 7.4 Hz,1H), 7.51 (d, J=7.3 Hz, 1H), 7.47-7.39 (m, 5H), 5.11-5.06 (m, 1H), 4.75(s, 2H), 4.68 (dd, J=8.8, 5.5 Hz, 1H), 3.47-3.31 (m, 5H), 2.83-2.65 (m,7H), 2.44 (s, 3H), 2.13-2.06 (m, 1H), 1.68 (s, 3H), 1.67-1.60 (m, 4H).¹³C NMR (100 MHz, cd₃od) δ 174.43, 172.40, 171.29, 169.92, 168.24,167.82, 166.71, 156.31, 153.14, 138.38, 138.24, 137.54, 134.88, 133.86,133.44, 132.29, 132.00, 131.49, 129.88, 122.46, 121.90, 119.38, 118.02,69.59, 54.96, 50.55, 40.09, 39.84, 38.45, 32.14, 27.75, 27.65, 23.62,14.41, 12.96, 11.56. MS 785.48 (M+H).

Example 3: Synthesis of dBET3

A 0.1 M solution ofN-(2-aminoethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.475 mL, 0.0475 mmol, 1.2 eq) was added toJQ-acid (15.86 mg, 0.0396 mmol, 1 eq) at room temperature. DIPEA (20.7microliters, 0.1188 mmol, 3 eq) and HATU (16.5 mg, 0.0435 mmol, 1.1 eq)were then added and the mixture was stirred for 24 hours, then purifiedby preparative HPLC to give a yellow solid (22.14 mg, 0.0292 mmol, 74%).¹H NMR (400 MHz, Methanol-d₄) δ 7.82-7.75 (m, 1H), 7.52-7.32 (m, 6H),5.04 (dd, J=11.6, 5.5 Hz, 1H), 4.76 (d, J=3.2 Hz, 2H), 4.66 (d, J=6.6Hz, 1H), 3.58-3.35 (m, 6H), 2.78-2.58 (m, 6H), 2.48-2.41 (m, 3H),2.11-2.02 (m, 1H), 1.70 (d, J=11.8 Hz, 3H). ¹³C NMR (100 MHz, cd₃od) δ174.38, 171.26, 171.19, 170.26, 168.86, 168.21, 167.76, 166.72, 156.27,153.14, 138.44, 138.36, 138.19, 134.87, 133.71, 132.31, 131.57, 131.51,129.90, 129.86, 121.81, 119.36, 117.95, 69.48, 54.83, 50.52, 40.09,39.76, 38.30, 32.09, 23.63, 14.40, 11.61. LCMS 757.41 (M+H).

Example 4: Synthesis of dBET5

A 0.1M solution ofN-(6-aminohexyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.247 mL, 0.0247 mmol, 1 eq) was added toJQ-acid (9.9 mg, 0.0247 mmol, 1 eq) at room temperature. DIPEA (12.9microliters, 0.0741 mmol, 3 eq) and HATU (9.4 mg, 0.0247 mmol, 1 eq)were then added. the mixture was stirred for 21 hours, then diluted withMeOH and concentrated under reduced pressure. The crude material waspurified by preparative HPLC to give a yellow solid (13.56 mg, 0.0167mmol, 67%). ¹H NMR (400 MHz, Methanol-d₄) δ 7.82-7.78 (m, 1H), 7.53 (dd,J=7.3, 2.0 Hz, 1H), 7.49-7.37 (m, 5H), 5.10 (dt, J=12.4, 5.3 Hz, 1H),4.76 (s, 2H), 4.70 (dd, J=8.7, 5.5 Hz, 1H), 3.42-3.33 (m, 2H), 3.25 (dt,J=12.3, 6.0 Hz, 3H), 2.87-2.67 (m, 7H), 2.48-2.42 (m, 3H), 2.14-2.09 (m,1H), 1.69 (d, J=4.8 Hz, 3H), 1.58 (s, 4H), 1.42 (d, J=5.2 Hz, 4H). ¹³CNMR (100 MHz, cd₃od) δ 174.51, 171.31, 171.26, 169.82, 168.27, 168.26,167.75, 156.26, 150.46, 138.20, 134.92, 133.92, 133.47, 132.34, 132.01,131.52, 129.88, 121.69, 119.34, 117.95, 111.42, 69.39, 54.97, 50.56,40.39, 40.00, 38.40, 32.15, 30.46, 30.16, 27.58, 27.48, 23.64, 14.41,12.96, 11.55. LCMS 813.38.

Example 5: Synthesis of dBET6

A 0.1M solution ofN-(8-aminooctyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.191 mL, 0.0191 mmol, 1 eq) was added toJQ-acid (7.66 mg, 0.0191 mmol, 1 eq) at room temperature. DIPEA (10microliters, 0.0574 mmol, 3 eq) and HATU (7.3 mg, 0.0191 mmol, 1 eq)were added and the mixture was stirred for 22 hours, diluted with MeOH,and concentrated under reduced pressure. The crude material was purifiedby preparative HPLC to give a cream colored solid. (8.53 mg, 0.0101mmol, 53%). ¹H NMR (400 MHz, Methanol-d₄) δ 7.80 (dd, J=8.4, 7.4 Hz,1H), 7.53 (d, J=7.4 Hz, 1H), 7.49-7.36 (m, 5H), 5.10 (dt, J=12.3, 5.3Hz, 1H), 4.75 (s, 2H), 4.69 (dd, J=8.8, 5.3 Hz, 1H), 3.42 (dd, J=15.0,8.9 Hz, 1H), 3.30-3.18 (m, 4H), 2.90-2.64 (m, 7H), 2.45 (s, 3H), 2.13(dtt, J=10.8, 5.2, 2.6 Hz, 1H), 1.71 (d, J=4.4 Hz, 3H), 1.56 (d, J=6.2Hz, 4H), 1.33 (d, J=17.1 Hz, 8H). ¹³C NMR (100 MHz, cd₃od) δ 174.50,172.38, 171.30, 169.81, 168.28, 167.74, 166.64, 156.25, 138.38, 138.20,137.55, 134.92, 133.88, 133.42, 132.27, 132.02, 131.50, 129.85, 121.66,119.30, 117.95, 69.37, 55.01, 50.58, 40.51, 40.12, 38.44, 32.18, 30.46,30.33, 30.27, 30.21, 27.91, 27.81, 23.63, 14.42, 12.96, 11.55. LCMS841.64 (M+H).

Example 6: Synthesis of dBET9

A 0.1M solution ofN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.321 mL, 0.0321 mmol, 1 eq) was added toJQ-acid (12.87 mg, 0.0321 mmol, 1 eq) at room temperature. DIPEA (16.8microliters, 0.0963 mmol, 3 eq) and HATU (12.2 mg, 0.0321 mmol, 1 eq)were added and the mixture was stirred for 24 hours, diluted with MeOH,and concentrated under reduced pressure. The crude material was purifiedby preparative HPLC to give a yellow oil. (16.11 mg, 0.0176 mmol, 55%).

¹H NMR (400 MHz, Methanol-d₄) δ 7.79 (dd, J=8.4, 7.4 Hz, 1H), 7.52 (d,J=7.2 Hz, 1H), 7.49-7.36 (m, 5H), 5.10 (dd, J=12.5, 5.5 Hz, 1H),4.78-4.67 (m, 3H), 3.64-3.52 (m, 11H), 3.48-3.32 (m, 6H), 2.94-2.64 (m,7H), 2.52-2.43 (m, 3H), 2.18-2.08 (m, 1H), 1.81 (p, J=6.3 Hz, 4H),1.73-1.67 (m, 3H). LCMS 918.45 (M+H).

Example 7: Synthesis of dBET17

A 0.1 M solution ofN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.281 mL, 0.0281 mmol 1 eq) was added to(S)-2-(4-(4-cyanophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)aceticacid (11 mg, 0.0281 mmol, 1 eq) at room temperature. DIPEA (14.7microliters, 0.0843 mmol, 3 eq) and HATU (10.7 mg, 0.0281 mmol, 1 eq)were added and the mixture was stirred for 24 hours, diluted with EtOAcand washed with saturated sodium bicarbonate, water and brine. Theorganic layer was dried over sodium sulfate, filtered and condensed.Purification by column chromatography (ISCO, 4 g silica column 0-10%MeOH/DCM) gave a white solid (14.12 mg, 0.0182 mmol, 65%).

¹H NMR (400 MHz, Methanol-d₄) δ 7.82-7.72 (m, 3H), 7.61 (dd, J=8.5, 2.0Hz, 2H), 7.51 (d, J=7.9 Hz, 1H), 7.44-7.40 (m, 1H), 5.11-5.05 (m, 1H),4.76 (s, 2H), 4.66 (dd, J=9.0, 5.1 Hz, 1H), 3.48-3.32 (m, 4H), 3.30-3.23(m, 1H), 2.87-2.61 (m, 7H), 2.43 (s, 3H), 2.10 (dt, J=10.7, 5.2 Hz, 1H),1.70-1.59 (m, 7H). ¹³C NMR (100 MHz, cd₃od) δ 174.42, 172.65, 171.27,169.92, 168.25, 167.80, 165.88, 156.31, 143.55, 138.24, 134.88, 133.92,133.50, 133.39, 131.72, 131.46, 130.55, 121.93, 119.39, 119.21, 118.02,115.17, 69.59, 55.50, 50.55, 40.10, 39.83, 38.86, 32.11, 27.78, 27.67,23.62, 14.41, 12.91, 11.64. LCMS 776.39 (M+H).

Example 8: Synthesis of dBET15

N-(6-aminohexyl)-2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-carboxamidetrifluoroacetate (13.29 mg, 0.258 mmol, 1 eq) and JQ-acid (10.3 mg,0.0258 mmol, 1 eq) were dissolved in DMF (0.26 mL). DIPEA (13.5microliters, 0.0775 mmol, 3 eq) was added, followed by HATU (9.8 mg,0.0258 mmol, 1 eq) and the mixture was stirred at room temperature.After 24 hours, the material was diluted with DCM and purified by columnchromatography (ISCO, 0-15% MeOH/DCM) followed by preparative HPLC togive a pale yellow solid (11.44 mg, 0.0146 mmol 57%).

¹H NMR (400 MHz, Methanol-d₄) δ 8.29-8.23 (m, 2H), 7.93 (dd, J=8.1, 4.2Hz, 1H), 7.50-7.34 (m, 4H), 5.17-5.11 (m, 1H), 4.75-4.69 (m, 1H),3.53-3.32 (m, 6H), 3.25 (dd, J=13.8, 6.7 Hz, 1H), 2.90-2.67 (m, 6H),2.49-2.38 (m, 3H), 2.18-2.10 (m, 1H), 1.64 (d, J=22.4 Hz, 6H), 1.47 (s,4H). ¹³C NMR (100 MHz, cd₃od) δ 174.48, 171.17, 168.05, 168.03, 167.99,167.70, 166.63, 141.81, 138.40, 137.47, 135.09, 134.77, 134.74, 133.96,133.94, 133.38, 132.24, 132.05, 131.44, 129.85, 124.57, 123.12, 123.09,54.98, 50.78, 40.88, 40.08, 38.37, 32.13, 30.40, 30.23, 27.34, 27.26,23.58, 14.40, 12.96, 11.54. LCMS 783.43 (M+H).

Example 9: Synthesis of dBET2

(1) Synthesis of (R)-ethyl4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoate

(R)-2-chloro-8-cyclopentyl-7-ethyl-5-methyl-7,8-dihydropteridin-6(5H)-one(44.2 mg, 0.15 mmol, 1 eq), ethyl 4-amino-3-methoxybenzoate (35.1 mg,0.18 mmol, 1.2 eq), Pd₂dba₃ (6.9 mg, 0.0075 mmol, 5 mol %), XPhos (10.7mg, 0.0225 mmol, 15 mol %) and potassium carbonate (82.9 mg, 0.60 mmol,4 eq) were dissolved in tBuOH (1.5 mL, 0.1 M) and heated to 100° C.After 21 hours, the mixture was cooled to room temperature, filteredthrough celite, washed with DCM and concentrated under reduced pressure.Purification by column chromatography (ISCO, 4 g silica column, 0-100%EtOAc/hexanes over an 18 minute gradient) gave a yellow oil (52.3 mg,0.115 mmol, 77%). ¹H NMR (400 MHz, Chloroform-d) δ 8.57 (d, J=8.5 Hz,1H), 7.69 (td, J=6.2, 2.9 Hz, 2H), 7.54 (d, J=1.8 Hz, 1H), 4.52 (t,J=7.9 Hz, 1H), 4.37 (q, J=7.1 Hz, 2H), 4.23 (dd, J=7.9, 3.7 Hz, 1H),3.97 (s, 3H), 3.33 (s, 3H), 2.20-2.12 (m, 1H), 2.03-1.97 (m, 1H), 1.86(ddd, J=13.9, 7.6, 3.6 Hz, 4H), 1.78-1.65 (m, 4H), 1.40 (t, J=7.1 Hz,3H), 0.88 (t, J=7.5 Hz, 3H). LCMS 454.32 (M+H).

(2) Synthesis of(R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoicacid

(R)-ethyl4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoate(73.8 mg, 0.163 mmol, 1 eq) and LiOH (11.7 mg, 0.489 mmol, 3 eq) weredissolved in MeOH (0.82 mL) THE (1.63 mL) and water (0.82 mL). After 20hours, an additional 0.82 mL of water was added and the mixture wasstirred for an additional 24 hours before being purified by preparativeHPLC to give a cream colored solid (53 mg, 0.125 mmol, 76%). ¹H NMR (400MHz, Methanol-d₄) δ 7.97 (d, J=8.4 Hz, 1H), 7.67 (dd, J=8.3, 1.6 Hz,1H), 7.64-7.59 (m, 2H), 4.38 (dd, J=7.0, 3.2 Hz, 1H), 4.36-4.29 (m, 1H),3.94 (s, 3H), 3.30 (s, 3H), 2.13-1.98 (m, 2H), 1.95-1.87 (m, 2H),1.87-1.76 (m, 2H), 1.73-1.57 (m, 4H), 0.86 (t, J=7.5 Hz, 3H). ¹³C NMR(100 MHz, cd₃od) δ 168.67, 163.72, 153.59, 150.74, 150.60, 130.95,127.88, 125.97, 123.14, 121.68, 116.75, 112.35, 61.76, 61.66, 56.31,29.40, 29.00, 28.68, 28.21, 23.57, 23.41, 8.69. LCMS 426.45 (M+H).

(3) Synthesis of dBET2

A 0.1 M solution ofN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.183 mL, 0.0183 mmol 1.2 eq) was added to(R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoicacid (6.48 mg, 0.0152 mmol, 1 eq) at room temperature. DIPEA (7.9microliters, 0.0456 mmol, 3 eq) and HATU (6.4 mg, 0.0168 mmol, 1.1 eq)were added and the mixture was stirred for 23 hours, before beingpurified by preparative HPLC to give a yellow solid (9.44 mg, 0.0102mmol, 67%). ¹H NMR (400 MHz, Methanol-d₄) δ 7.84-7.77 (m, 2H), 7.58 (d,J=1.8 Hz, 2H), 7.53-7.46 (m, 2H), 7.42 (d, J=8.4 Hz, 1H), 5.11-5.05 (m,1H), 4.76 (s, 2H), 4.48 (dd, J=6.5, 3.1 Hz, 1H), 4.33-4.24 (m, 1H), 3.95(s, 3H), 3.49-3.35 (m, 4H), 2.97 (d, J=10.5 Hz, 3H), 2.89-2.65 (m, 5H),2.17-1.99 (m, 4H), 1.89 (dd, J=14.5, 7.3 Hz, 2H), 1.69-1.54 (m, 6H),1.36 (dt, J=7.6, 3.9 Hz, 1H), 0.85 (t, J=7.5 Hz, 3H). ¹³C NMR (100 MHz,cd₃od) δ 176.52, 174.48, 173.05, 171.34, 169.99, 168.91, 168.25, 167.80,164.58, 156.34, 154.48, 153.10, 150.63, 138.22, 134.89, 133.96, 129.53,123.93, 121.87, 120.78, 119.36, 117.99, 111.54, 69.55, 63.29, 63.10,56.68, 50.55, 40.71, 39.86, 32.15, 29.43, 29.26, 28.73, 28.63, 27.81,27.77, 24.25, 23.63, 8.47. LCMS 810.58 (M+H).

Example 10: Synthesis of dBET7

A 0.1 M solutionN-(6-aminohexyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.186 mL, 0.0186 mmol 1 eq) was added to(R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoicacid (7.9 mg, 0.0186 mmol, 1 eq) at room temperature. DIPEA (9.7microliters, 0.0557 mmol, 3 eq) and HATU (7.1 mg, 0.0186 mmol, 1 eq)were added and the mixture was stirred for 19 hours, before beingpurified by preparative HPLC to give the desired trifluoracetate salt asa yellow solid (13.62 mg, 0.0143 mmol, 77%).

¹H NMR (400 MHz, Methanol-d₄) δ 7.80 (t, J=8.3 Hz, 2H), 7.61-7.57 (m,2H), 7.55-7.49 (m, 2H), 7.42 (d, J=8.4 Hz, 1H), 5.13 (dd, J=12.6, 5.5Hz, 1H), 4.75 (s, 2H), 4.48 (dd, J=6.5, 3.2 Hz, 1H), 4.33-4.24 (m, 1H),3.97 (s, 3H), 3.40 (t, J=7.1 Hz, 2H), 3.34 (d, J=6.7 Hz, 2H), 3.30 (s,3H), 2.98 (d, J=8.5 Hz, 1H), 2.89-2.82 (m, 1H), 2.79-2.63 (m, 3H),2.17-2.00 (m, 4H), 1.91 (dt, J=14.4, 7.1 Hz, 3H), 1.61 (dt, J=13.4, 6.6Hz, 7H), 1.47-1.41 (m, 3H), 0.86 (t, J=7.5 Hz, 3H). ¹³C NMR (100 MHz,cd₃od) δ 174.54, 171.37, 169.84, 168.84, 168.27, 167.74, 164.59, 156.26,154.47, 153.18, 150.69, 138.19, 134.91, 134.05, 129.47, 124.78, 124.01,121.65, 120.77, 119.29, 117.92, 117.86, 111.55, 69.34, 63.31, 63.13,56.67, 50.53, 40.97, 39.96, 32.16, 30.42, 30.19, 29.42, 29.26, 28.72,28.62, 27.65, 27.46, 24.26, 23.65, 8.47. LCMS 838.60 (M+H).

Example 11: Synthesis of dBET8

A 0.1 M solutionN-(8-aminooctyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.186 mL, 0.0186 mmol 1 eq) was added to(R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoicacid (7.9 mg, 0.0186 mmol, 1 eq) at room temperature. DIPEA (9.7microliters, 0.0557 mmol, 3 eq) and HATU (7.1 mg, 0.0186 mmol, 1 eq)were added and the mixture was stirred for 16 hours, before beingpurified by preparative HPLC to give the desired trifluoracetate salt asan off-white solid (7.15 mg, 0.007296 mmol, 39%).

¹H NMR (400 MHz, Methanol-d₄) δ 7.83-7.77 (m, 2H), 7.61-7.56 (m, 2H),7.55-7.50 (m, 2H), 7.42 (d, J=8.5 Hz, 1H), 5.13 (dd, J=12.6, 5.5 Hz,1H), 4.75 (s, 2H), 4.49 (dd, J=6.6, 3.3 Hz, 1H), 4.33-4.24 (m, 1H), 3.97(s, 3H), 3.39 (t, J=7.1 Hz, 2H), 3.34-3.32 (m, 2H), 3.30 (s, 3H),3.01-2.83 (m, 2H), 2.82-2.65 (m, 3H), 2.17-2.01 (m, 4H), 1.91 (dt,J=14.2, 7.4 Hz, 1H), 1.68-1.54 (m, 7H), 1.37 (s, 7H), 0.86 (t, J=7.5 Hz,3H). ¹³C NMR (100 MHz, cd₃od) δ 174.52, 171.35, 169.81, 168.85, 168.28,167.74, 164.58, 156.27, 154.47, 153.89, 150.64, 138.19, 134.93, 134.18,129.52, 129.41, 124.91, 123.83, 121.67, 120.76, 119.31, 117.95, 117.89,111.57, 69.37, 63.37, 63.17, 56.67, 50.58, 41.12, 40.12, 32.19, 30.43,30.28, 30.22, 30.19, 29.40, 29.25, 28.71, 28.62, 27.94, 27.75, 24.29,23.65, 8.46. LCMS 866.56 (M+H).

Example 12: Synthesis of dBET10

A 0.1 M solutionN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.172 mL, 0.0172 mmol 1 eq) was added to(R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoicacid (7.3 mg, 0.0172 mmol, 1 eq) at room temperature. DIPEA (9.0microliters, 0.0515 mmol, 3 eq) and HATU (6.5 mg, 0.0172 mmol, 1 eq)were added and the mixture was stirred for 23 hours, before beingpurified by preparative HPLC to give the desired trifluoracetate salt asan off-white oil (10.7 mg, 0.0101 mmol, 59%). ¹H NMR (400 MHz,Methanol-d₄) δ 7.78 (d, J=8.3 Hz, 1H), 7.75 (dd, J=8.4, 7.4 Hz, 1H),7.56-7.51 (m, 2H), 7.49-7.44 (m, 2H), 7.36 (d, J=8.4 Hz, 1H), 5.08 (dd,J=12.4, 5.4 Hz, 1H), 4.69 (s, 2H), 4.44 (dd, J=6.7, 3.2 Hz, 1H),4.30-4.21 (m, 1H), 3.92 (s, 3H), 3.59-3.42 (m, 12H), 3.35 (t, J=6.7 Hz,2H), 3.25 (s, 3H), 2.95-2.64 (m, 5H), 2.13-1.95 (m, 4H), 1.91-1.71 (m,7H), 1.65-1.48 (m, 4H), 0.81 (t, J=7.5 Hz, 3H). ¹³C NMR (100 MHz, cd₃od)δ 174.50, 171.35, 169.83, 168.77, 168.25, 167.68, 164.57, 156.26,154.47, 153.05, 150.59, 138.19, 134.92, 133.89, 129.53, 124.57, 123.98,121.72, 120.75, 119.26, 117.95, 117.86, 111.54, 71.51, 71.46, 71.28,71.20, 70.18, 69.65, 69.41, 63.27, 63.07, 56.71, 50.57, 38.84, 37.59,32.17, 30.41, 30.32, 29.46, 29.26, 28.73, 28.64, 24.27, 23.65, 8.49.LCMS 942.62 (M+H).

Example 13: Synthesis of dBET16

A 0.1 M solution ofN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.402 mL, 0.0402 mmol 1 eq) was added(R)-4-((4-cyclopentyl-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazin-6-yl)amino)-3-methoxybenzoicacid (16.55 mg, 0.0402 mmol, 1 eq) at room temperature. DIPEA (21microliters, 0.1206 mmol, 3 eq) and HATU (15.3 mg, 0.0402 mmol, 1 eq)were added and the mixture was stirred for 21 hours, before beingpurified by preparative HPLC, followed by column chromatography (ISCO,12 g NH2-silica column, 0-15% MeOH/DCM, 20 min gradient) to give HPLC togive a brown solid (10.63 mg, 0.0134 mmol, 33%). ¹H NMR (400 MHz,Methanol-d₄) δ 8.22 (d, J=8.4 Hz, 1H), 7.78 (dd, J=8.4, 7.4 Hz, 1H),7.73-7.68 (m, 1H), 7.49 (d, J=7.4 Hz, 2H), 7.46-7.39 (m, 2H), 6.98 (d,J=8.8 Hz, 1H), 5.97-5.87 (m, 1H), 5.06 (dd, J=12.6, 5.4 Hz, 1H), 4.76(s, 2H), 3.98 (s, 3H), 3.61 (s, 2H), 3.44-3.36 (m, 4H), 2.92 (s, 1H),2.78 (dd, J=14.3, 5.2 Hz, 1H), 2.68 (ddd, J=17.7, 8.2, 4.5 Hz, 2H),2.36-2.26 (m, 2H), 2.10-1.90 (m, 5H), 1.76-1.62 (m, 6H), 1.31 (d, J=16.0Hz, 4H). LCMS 795.38 (M+H).

Example 14: Synthesis of dBET11

(1) Synthesis of ethyl4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-methoxybenzoate

2-chloro-5,11-dimethyl-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one(82.4 mg, 0.30 mmol, 1 eq), ethyl 4-amino-3-methoxybenzoate (70.3 mg,0.36 mmol, 1.2 eq) Pd₂dba₃ (13.7 mg, 0.015 mmol, 5 mol %), XPhos (21.5mg, 0.045 mmol, 15 mol %) and potassium carbonate (166 mg, 1.2 mmol, 4eq) were dissolved in tBuOH (3.0 mL) and heated to 100° C. After 17hours, the mixture was cooled room temperature and filtered throughcelite. The mixture was purified by column chromatography (ISCO, 12 gsilica column, 0-100% EtOAc/hexanes, 19 min gradient) to give an offwhite solid (64.3 mg, 0.148 mmol, 49%).

¹H NMR (400 MHz, 50% cd₃od/cdcl₃) δ 8.51 (d, J=8.5 Hz, 1H), 8.17 (s,1H), 7.73 (ddd, J=18.7, 8.1, 1.7 Hz, 2H), 7.52 (d, J=1.8 Hz, 1H),7.46-7.41 (m, 1H), 7.15-7.10 (m, 2H), 4.34 (q, J=7.1 Hz, 4H), 3.95 (s,3H), 3.47 (s, 3H), 3.43 (s, 3H), 1.38 (t, J=7.1 Hz, 3H). ¹³C NMR (100MHz, 50% cd₃od/cdcl₃) δ 169.28, 167.39, 164.29, 155.64, 151.75, 149.73,147.45, 146.22, 133.88, 133.18, 132.37, 126.44, 124.29, 123.70, 123.36,122.26, 120.58, 118.05, 116.83, 110.82, 61.34, 56.20, 38.62, 36.25,14.51. LCMS 434.33 (M+H).

(2) Synthesis of4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-methoxybenzoicacid

Ethyl4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-methoxybenzoate(108.9 mg, 0.251 mmol, 1 eq) and LiGH (18 mg) were dissolved in THE (2.5mL) and water (1.25 mL). After 24 hours, MeOH (0.63 mL) was added toimproved solubility) and stirred for an additional 24 hours before beingdiluted with MeOH and purified by preparative HPLC to give a lightyellow solid (41.31 mg).

¹H NMR (400 MHz, Methanol-d₄) δ 8.51 (d, J=8.5 Hz, 1H), 8.22 (s, 1H),7.73 (ddd, J=11.8, 8.1, 1.7 Hz, 2H), 7.57 (d, J=1.8 Hz, 1H), 7.49-7.44(m, 1H), 7.19-7.11 (m, 2H), 3.97 (s, 3H), 3.48 (s, 3H), 3.45 (s, 3H).LCMS 406.32 (M+H).

(3) Synthesis of dBET11

A 0.1 M solution ofN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.190 mL, 0.0190 mmol 1 eq) was added to4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-methoxybenzoicacid (7.71 mg, 0.0190 mmol, 1 eq) at room temperature. DIPEA (9.9microliters, 0.0571 mmol, 3 eq) and HATU (7.2 mg, 0.0190 mmol, 1 eq)were added and the mixture was stirred for 22 hours, before beingpurified by preparative HPLC to give HPLC to give the desiredtrifluoracetate salt as a cream colored solid (6.72 mg, 0.00744 mmol,39%). ¹H NMR (400 MHz, Methanol-d₄) δ 8.46 (d, J=8.3 Hz, 1H), 8.21 (s,1H), 7.79-7.73 (m, 2H), 7.52 (d, J=7.1 Hz, 1H), 7.50-7.43 (m, 3H), 7.33(d, J=8.2 Hz, 1H), 7.15 (dd, J=7.7, 5.9 Hz, 2H), 4.98 (dd, J=12.0, 5.5Hz, 1H), 4.69 (s, 2H), 3.97 (s, 3H), 3.49 (s, 3H), 3.46-3.34 (m, 7H),2.81-2.67 (m, 3H), 2.13-2.08 (m, 1H), 1.69 (dt, J=6.6, 3.5 Hz, 4H). 13CNMR (100 MHz, cd₃od) δ 173.40, 170.10, 169.68, 169.00, 168.85, 167.60,167.15, 164.77, 156.01, 155.42, 151.83, 150.03, 148.21, 137.82, 134.12,133.48, 132.58, 132.52, 128.11, 126.72, 124.54, 122.33, 121.06, 120.63,118.77, 118.38, 117.94, 117.62, 109.67, 68.90, 56.33, 49.96, 40.16,39.48, 38.72, 36.34, 31.82, 27.24, 23.16. LCMS 790.48 (M+H).

Example 15: Synthesis of dBET12

A 0.1M solutionN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetatein DMF (0.186 mL, 0.0186 mmol 1 eq) was added to4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-methoxybenzoicacid (7.53 mg, 0.0186 mmol, 1 eq) at room temperature. DIPEA (9.7microliters, 0.0557 mmol, 3 eq) and HATU (7.1 mg, 0.0186 mmol, 1 eq)were added and the mixture was stirred for 22 hours, before beingpurified by preparative HPLC to give HPLC to give the desiredtrifluoracetate salt as a cream colored solid (7.50 mg, 0.00724 mmol,39%).

¹H NMR (400 MHz, Methanol-d₄) δ 8.46 (d, J=8.9 Hz, 1H), 8.21 (s, 1H),7.73 (dd, J=15.2, 7.8 Hz, 2H), 7.50-7.42 (m, 3H), 7.28 (d, J=8.5 Hz,1H), 7.15 (t, J=7.7 Hz, 2H), 5.01 (dd, J=11.8, 5.8 Hz, 1H), 4.68 (s,2H), 3.97 (s, 3H), 3.67-3.58 (m, 7H), 3.58-3.43 (m, 10H), 3.39 (t, J=6.8Hz, 2H), 3.35 (s, 2H), 2.97 (s, 1H), 2.84-2.70 (m, 3H), 2.16-2.07 (m,1H), 1.93-1.76 (m, 4H). LCMS 922.57 (M+H).

Example 16: Synthesis of dBET13

A 0.1 M solution ofN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.501 mL, 0.0501 mmol 1 eq) was added to2-((2-(4-(3,5-dimethylisoxazol-4-yl)phenyl)imidazo[1,2-a]pyrazin-3-yl)amino)aceticacid (synthesized as in McKeown et al, J. Med. Chem, 2014, 57, 9019)(18.22 mg, 0.0501 mmol, 1 eq) at room temperature. DIPEA (26.3microliters, 0.150 mmol, 3 eq) and HATU (19.0 mg, 0.0501 mmol, 1 eq)were added and the mixture was stirred for 21 hours, before beingpurified by preparative HPLC to give HPLC to give the desiredtrifluoracetate salt as a dark yellow oil (29.66 mg, 0.0344 mmol, 69%).¹H NMR (400 MHz, Methanol-d₄) δ 9.09 (s, 1H), 8.65 (d, J=5.2 Hz, 1H),8.14-8.06 (m, 2H), 7.94-7.88 (m, 1H), 7.80-7.74 (m, 1H), 7.59-7.47 (m,3H), 7.40 (dd, J=8.4, 4.7 Hz, 1H), 5.11-5.06 (m, 1H), 4.72 (d, J=9.8 Hz,2H), 3.90 (s, 2H), 3.25-3.22 (m, 1H), 3.12 (t, J=6.4 Hz, 1H), 2.96 (s,2H), 2.89-2.79 (m, 1H), 2.76-2.62 (m, 2H), 2.48-2.42 (m, 3H), 2.29 (s,3H), 2.10 (ddq, J=10.2, 5.3, 2.7 Hz, 1H), 1.49-1.45 (m, 2H), 1.37 (dd,J=6.7, 3.6 Hz, 2H). ¹³C NMR (100 MHz, cd₃od) δ 174.45, 171.98, 171.35,169.88, 168.17, 167.85, 167.40, 159.88, 156.28, 141.82, 138.26, 135.85,134.82, 133.09, 132.06, 130.75, 129.67, 122.07, 121.94, 119.30, 118.98,118.06, 117.24, 69.56, 50.56, 40.05, 39.73, 32.13, 27.53, 23.62, 18.71,17.28, 11.64, 10.85. LCMS 748.49 (M+H).

Example 17: Synthesis of dBET14

A 0.1 M solutionN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate in DMF (0.510 mL, 0.0510 mmol 1 eq) was added to2-((2-(4-(3,5-dimethylisoxazol-4-yl)phenyl)imidazo[1,2-a]pyrazin-3-yl)amino)aceticacid (synthesized as in McKeown et al, J. Med. Chem, 2014, 57, 9019)(18.52 mg, 0.0510 mmol, 1 eq) at room temperature. DIPEA (26.6microliters, 0.153 mmol, 3 eq) and HATU (19.4 mg, 0.0510 mmol, 1 eq)were added and the mixture was stirred for 22 hours, before beingpurified by preparative HPLC to give HPLC to give the desiredtrifluoracetate salt as a dark yellow oil (32.63 mg, 0.0328 mmol, 64%).

¹H NMR (400 MHz, Methanol-d₄) δ 9.09 (s, 1H), 8.66 (d, J=5.4 Hz, 1H),8.17-8.08 (m, 2H), 7.92 (d, J=5.6 Hz, 1H), 7.77 (dd, J=8.4, 7.4 Hz, 1H),7.60-7.47 (m, 3H), 7.39 (d, J=8.4 Hz, 1H), 5.09 (dd, J=12.4, 5.5 Hz,1H), 4.71 (s, 2H), 3.91 (s, 2H), 3.62-3.46 (m, 10H), 3.38 (dt, J=16.0,6.4 Hz, 3H), 3.18 (t, J=6.8 Hz, 2H), 2.97 (s, 1H), 2.89-2.81 (m, 1H),2.78-2.66 (m, 2H), 2.47 (s, 3H), 2.31 (s, 3H), 2.16-2.08 (m, 1H), 1.79(dt, J=12.8, 6.5 Hz, 2H), 1.64 (t, J=6.3 Hz, 2H). ¹³C NMR (100 MHz,cd₃od) δ 174.48, 171.88, 171.34, 169.80, 168.22, 167.69, 167.42, 159.87,156.24, 141.87, 138.21, 135.89, 134.88, 133.13, 132.04, 130.76, 129.67,122.08, 121.69, 119.20, 117.94, 117.23, 71.44, 71.22, 71.10, 69.92,69.62, 69.38, 50.57, 49.64, 38.11, 37.55, 32.16, 30.30, 30.20, 23.63,11.67, 10.88. LCMS 880.46 (M+H).

Example 18: Synthesis of dBET18

(1) Synthesis of (S)-tert-butyl4-(3-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)propyl)piperazine-1-carboxylate

JQ-acid (176.6 mg, 0.441 mmol, 1 eq) was dissolved in DMF (4.4 mL) atroom temperature. HATU (176 mg, 0.463 mmol, 1.05 eq) was added, followedby DIPEA (0.23 mL), 1.32 mmol, 3 eq). After 10 minutes, tert-butyl4-(3-aminopropyl)piperazine-1-carboxylate (118 mg, 0.485 mmol, 1.1 eq)was added as a solution in DMF (0.44 mL). After 24 hours, the mixturewas diluted with half saturated sodium bicarbonate and extracted twicewith DCM and once with EtOAc. The combined organic layer was dried oversodium sulfate, filtered and condensed. Purification by columnchromatography (ISCO, 24 g silica column, 0-15% MeOH/DCM, 23 minutegradient) gave a yellow oil (325.5 mg, quant yield)

¹H NMR (400 MHz, Chloroform-d) δ 7.67 (t, J=5.3 Hz, 1H), 7.41-7.28 (m,4H), 4.58 (dd, J=7.5, 5.9 Hz, 1H), 3.52-3.23 (m, 8H), 2.63 (s, 9H), 2.37(s, 3H), 1.80-1.69 (m, 2H), 1.64 (s, 3H), 1.42 (s, 9H). ¹³C NMR (100MHz, cdcl₃) δ 171.41, 164.35, 155.62, 154.45, 150.20, 136.92, 136.64,132.19, 131.14, 130.98, 130.42, 129.98, 128.80, 80.24, 56.11, 54.32,52.70, 38.96, 37.85, 28.42, 25.17, 14.43, 13.16, 11.82. LCMS 626.36(M+H).

(2) Synthesis of(S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(3-(piperazin-1-yl)propyl)acetamide

(S)-tert-butyl4-(3-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)propyl)piperazine-1-carboxylate(325.5 mg) was dissolved in DCM (5 mL) and MeOH (0.5 mL). A solution of4M HCl in dioxane (1 mL) was added and the mixture was stirred for 16hours, then concentrated under a stream of nitrogen to give a yellowsolid (231.8 mg) which was used without further purification.

¹H NMR (400 MHz, Methanol-d₄) δ 7.64-7.53 (m, 4H), 5.05 (t, J=7.1 Hz,1H), 3.81-3.66 (m, 6H), 3.62-3.33 (m, 9H), 3.30 (p, J=1.6 Hz, 1H), 2.94(s, 3H), 2.51 (s, 3H), 2.09 (dq, J=11.8, 6.1 Hz, 2H), 1.72 (s, 3H). ¹³CNMR (100 MHz, cd₃od) δ 171.78, 169.38, 155.83, 154.03, 152.14, 140.55,136.33, 134.58, 134.53, 133.33, 132.73, 130.89, 130.38, 56.07, 53.54,41.96, 37.22, 36.23, 25.11, 14.48, 13.14, 11.68. LCMS 526.29 (M+H).

(3) Synthesis of (S)-tert-butyl(6-(4-(3-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)propyl)piperazin-1-yl)₀₋₆-oxohexyl)carbamate

(S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(3-(piperazin-1-yl)propyl)acetamide(62.1 mg) and 6-((tert-butoxycarbonyl)amino)hexanoic acid (24.0 mg,0.1037 mmol, 1 eq) were dissolved in DMF (1 mL). DIPEA (72.2microliters, 0.4147 mmol, 4 eq) was added, followed by HATU (39.4 mg,0.1037 mmol, 1 eq) and the mixture was stirred for 25 hours. The mixturewas diluted with half saturated sodium bicarbonate and extracted threetimes with DCM. The combined organic layer was dried over sodiumsulfate, filtered and condensed. Purification by column chromatography(ISCO, 4 g silica column, 0-15% MeOH/DCM, 15 minute gradient) gave ayellow oil (71.75 mg, 0.0970 mmol, 94%).

¹H NMR (400 MHz, Chloroform-d) δ 7.61 (s, 1H), 7.43-7.28 (m, 4H), 4.63(s, 1H), 4.61-4.56 (m, 1H), 3.82-3.21 (m, 10H), 3.11-3.01 (m, 2H), 2.61(d, J=24.3 Hz, 9H), 2.38 (s, 3H), 2.28 (t, J=7.4 Hz, 2H), 1.73 (dq,J=13.8, 7.4 Hz, 2H), 1.63-1.55 (m, 2H), 1.53-1.24 (m, 14H). ¹³C NMR (100MHz, cdc₃) δ 171.63, 171.11, 164.34, 156.17, 155.66, 150.21, 136.96,136.72, 132.25, 131.14, 131.01, 130.47, 130.00, 128.85, 79.11, 56.42,54.46, 53.06, 52.82, 45.04, 41.02, 40.47, 39.29, 38.33, 33.00, 29.90,28.54, 26.60, 25.29, 24.86, 14.47, 13.20, 11.86. LCMS 739.37 (M+H).

(4) Synthesis of(S)—N-(3-(4-(6-aminohexanoyl)piperazin-1-yl)propyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide

(S)-tert-butyl(6-(4-(3-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)propyl)piperazin-1-yl)₀₋₆-oxohexyl)carbamate(71.75 mg, 0.0970 mmol, 1 eq) was dissolved in DCM (2 mL) and MeOH (0.2mL). A solution of 4M HCl in dioxane (0.49 mL) was added and the mixturewas stirred for 2 hours, then concentrated under a stream of nitrogen,followed by vacuum to give a yellow foam (59.8 mg, 0.0840 mmol, 87%).

¹H NMR (400 MHz, Methanol-d₄) δ 7.68-7.53 (m, 4H), 5.04 (d, J=6.6 Hz,1H), 4.66 (d, J=13.6 Hz, 1H), 4.23 (d, J=13.6 Hz, 1H), 3.63-3.34 (m,7H), 3.29-3.00 (m, 5H), 2.95 (d, J=6.0 Hz, 5H), 2.51 (d, J=9.2 Hz, 5H),2.08 (s, 2H), 1.77-1.62 (m, 7H), 1.45 (dt, J=15.3, 8.6 Hz, 2H). ¹³C NMR(100 MHz, cd₃od) δ 173.77, 171.84, 169.35, 155.85, 153.99, 140.56,136.40, 134.58, 133.35, 132.70, 130.39, 55.83, 53.57, 52.92, 52.70,43.57, 40.55, 39.67, 37.33, 36.25, 33.17, 28.26, 26.94, 25.33, 25.26,14.49, 13.15, 11.65. LCMS 639.35 (M+H).

(5) Synthesis of dBET18

(S)—N-(3-(4-(6-aminohexanoyl)piperazin-1-yl)propyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamidedihydrochloride (20.0 mg, 0.0281 mmol, 1 eq) and2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid(9.32 mg, 0.0281 mmol, 1 eq) were dissolved in DMF (0.281 mL). DIPEA(19.6 microliters, 0.1124 mmol, 4 eq) was added, followed by HATU (10.7mg, 0.0281 mmol, 1 eq). After 24 hours, the mixture was diluted withMeOH and purified by preparative HPLC to give the desiredtrifluoracetate salt.

¹H NMR (400 MHz, Methanol-d₄) δ 7.83-7.79 (m, 1H), 7.54 (d, J=7.1 Hz,1H), 7.45 (q, J=8.8 Hz, 5H), 5.12 (dd, J=12.5, 5.4 Hz, 1H), 4.76 (s,2H), 4.68 (t, J=7.3 Hz, 1H), 3.59-3.32 (m, 8H), 3.28-3.18 (m, 4H), 2.87(ddd, J=19.0, 14.7, 5.3 Hz, 2H), 2.80-2.65 (m, 6H), 2.44 (d, J=6.8 Hz,5H), 2.33-2.25 (m, 1H), 2.14 (dd, J=9.8, 4.9 Hz, 1H), 2.06-1.89 (m, 3H),1.70 (s, 3H), 1.61 (dq, J=14.4, 7.3, 6.9 Hz, 4H), 1.45-1.37 (m, 2H). ¹³CNMR (100 MHz, cd₃od) δ 174.52, 173.97, 173.69, 171.44, 169.88, 168.26,167.83, 166.72, 156.36, 138.28, 137.84, 134.89, 133.52, 132.12, 131.83,131.38, 129.89, 121.87, 119.32, 118.01, 69.52, 55.64, 55.03, 52.79,50.58, 43.69, 39.77, 38.57, 36.89, 33.47, 32.16, 29.93, 27.34, 25.76,25.45, 23.63, 14.39, 12.94, 11.66. LCMS 953.43 (M+H).

Example 19: Synthesis of dGR1

Example 20: Synthesis of dGR2

Example 21: Synthesis of dGR3

Example 22: Synthesis of dFKBP-1

(1) Synthesis of SLF-succinate

SLF (25 mg, 2.5 mL of a 10 mg/mL solution in MeOAc, 0.0477 mmol, 1 eq)was combined with DMF (0.48 mL, 0.1 M) and succinic anhydride (7.2 mg,0.0715 mmol, 1.5 eq) and stirred at room temperature for 24 hours. Lowconversion was observed and the mixture was placed under a stream of N2to remove the MeOAc. An additional 0.48 mL of DMF was added, along withan additional 7.2 mg succinic anhydride and DMAP (5.8 mg, 0.0477 mmol, 1eq). The mixture was then stirred for an additional 24 hours beforebeing purified by preparative HPLC to give SLF-succinate as a yellow oil(24.06 mg, 0.0385 mmol, 81%).

¹H NMR (400 MHz, Methanol-d₄) δ 7.62 (d, J=10.7 Hz, 1H), 7.44 (d, J=8.0Hz, 1H), 7.26 (td, J=7.9, 2.7 Hz, 1H), 7.07-6.97 (m, 1H), 6.80 (dd,J=8.1, 2.1 Hz, 1H), 6.74-6.66 (m, 2H), 5.73 (dd, J=8.1, 5.5 Hz, 1H),5.23 (d, J=4.8 Hz, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.39-3.29 (m, 4H),3.21 (td, J=13.2, 3.0 Hz, 1H), 2.68-2.50 (m, 5H), 2.37-2.19 (m, 2H),2.12-2.02 (m, 1H), 1.79-1.61 (m, 4H), 1.49-1.30 (m, 2H), 1.27-1.05 (m,6H), 0.82 (dt, J=41.2, 7.5 Hz, 3H). LCMS 624.72 (M+H).

(2) Synthesis of dFKBP-1

N-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate (9.9 mg, 0.0192 mmol, 1 eq) was added to SLFsuccinate(11.98 mg, 0.0192 mmol, 1 eq) as a solution in 0.192 mL DMF (0.1 M).DIPEA (10.0 microliters, 0.0575 mmol, 3 eq) was added, followed by HATU(7.3 mg, 0.0192 mmol, 1 eq). The mixture was stirred for 17 hours, thendiluted with MeOH and purified by preparative HPLC to give dFKBP-1 (7.7mg, 0.00763 mmol, 40%) as a yellow solid.

¹H NMR (400 MHz, Methanol-d₄) δ 7.81 (s, 1H), 7.77-7.70 (m, 1H),7.55-7.49 (m, 2H), 7.26 (dd, J=8.0, 5.3 Hz, 2H), 7.05-6.99 (m, 1H), 6.77(d, J=8.8 Hz, 1H), 6.66 (d, J=6.8 Hz, 2H), 5.77-5.72 (m, 1H), 5.24 (d,J=4.8 Hz, 1H), 4.99 (dd, J=12.3, 5.7 Hz, 1H), 4.68-4.59 (m, 2H), 3.82(s, 3H), 3.81 (s, 3H), 3.32 (dt, J=3.3, 1.6 Hz, 4H), 3.26-3.14 (m, 3H),2.79 (dd, J=18.9, 10.2 Hz, 3H), 2.64-2.48 (m, 5H), 2.34 (d, J=14.4 Hz,1H), 2.22 (d, J=9.2 Hz, 1H), 2.14-2.02 (m, 2H), 1.78-1.49 (m, 9H),1.43-1.30 (m, 2H), 1.20-1.04 (m, 6H), 0.90-0.76 (m, 3H). 13C NMR (100MHz, cd₃od) δ 208.51, 173.27, 172.64, 171.63, 169.93, 169.51, 168.04,167.69, 167.09, 166.71, 154.92, 149.05, 147.48, 140.76, 138.89, 137.48,133.91, 133.67, 129.36, 122.19, 120.61, 120.54, 119.82, 118.41, 118.12,117.79, 112.12, 111.76, 68.54, 56.10, 55.98, 51.67, 46.94, 44.57, 39.32,39.01, 38.23, 32.64, 31.55, 31.43, 26.68, 26.64, 25.08, 23.52, 23.21,22.85, 21.27, 8.76. LCMS 1009.66 (M+H).

Example 23: Synthesis of dFKBP-2

(1) Synthesis of tert-butyl(1-chloro-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamate

tert-butyl (3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate (1.0g, 3.12 mmol, 1 eq) was dissolved in THE (31 mL, 0.1 M). DIPEA (0.543mL, 3.12 mmol, 1 eq) was added and the solution was cooled to 0° C.Chloroacetyl chloride (0.273 mL, 3.43 mmool, 1.1 eq) was added and themixture was warmed slowly to room temperature. After 24 hours, themixture was diluted with EtOAc and washed with saturated sodiumbicarbonate, water then brine. The organic layer was dried over sodiumsulfate, filtered and condensed to give a yellow oil (1.416 g) that wascarried forward without further purification.

¹H NMR (400 MHz, Chloroform-d) δ 7.24 (s, 1H), 5.00 (s, 1H), 3.98-3.89(m, 2H), 3.54 (dddt, J=17.0, 11.2, 5.9, 2.2 Hz, 10H), 3.47-3.40 (m, 2H),3.37-3.31 (m, 2H), 3.17-3.07 (m, 2H), 1.79-1.70 (m, 2H), 1.67 (p, J=6.1Hz, 2H), 1.35 (s, 9H). ¹³C NMR (100 MHz, cdcl₃) δ 165.83, 155.97, 78.75,70.49, 70.47, 70.38, 70.30, 70.14, 69.48, 42.61, 38.62, 38.44, 29.62,28.59, 28.40. LCMS 397.37 (M+H).

(2) Synthesis of dimethyl3-((2,2-dimethyl-4,20-dioxo-3,9,12,15-tetraoxa-5,19-diazahenicosan-21-yl)oxy)phthalate

tert-butyl (1-chloro-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamate(1.41 g, 3.12 mmol, 1 eq) was dissolved in MeCN (32 mL, 0.1 M). Dimethyl3-hydroxyphthalate (0.721 g, 3.43 mmol, 1.1 eq) and cesium carbonate(2.80 g, 8.58 mmol, 2.75 eq) were added. The flask was fitted with areflux condenser and heated to 80° C. for 19 hours. The mixture wascooled to room temperature and diluted water and extracted once withchloroform and twice with EtOAc. The combined organic layers were driedover sodium sulfate, filtered and concentrated under reduced pressure.The crude material was purified by column chromatography (ISCO, 24 gsilica column, 0-15% MeOH/DCM 22 minute gradient) to give a yellow oil(1.5892 g, 2.78 mmol, 89% over two steps).

¹H NMR (400 MHz, Chloroform-d) δ 7.52 (d, J=7.8 Hz, 1H), 7.35 (t, J=8.1Hz, 1H), 7.04 (d, J=8.3 Hz, 1H), 7.00 (t, J=5.3 Hz, 1H), 5.06 (s, 1H),4.46 (s, 2H), 3.83 (s, 3H), 3.78 (s, 3H), 3.47 (ddd, J=14.9, 5.5, 2.8Hz, 8H), 3.39 (dt, J=9.4, 6.0 Hz, 4H), 3.29 (q, J=6.5 Hz, 2H), 3.09 (d,J=6.0 Hz, 2H), 1.70 (p, J=6.5 Hz, 2H), 1.63 (p, J=6.3 Hz, 2H), 1.31 (s,9H). ¹³C NMR (100 MHz, cdcl₃) δ 167.68, 167.36, 165.45, 155.93, 154.41,130.87, 129.60, 125.01, 123.20, 117.06, 78.60, 70.40, 70.17, 70.06,69.39, 68.67, 68.25, 52.77, 52.57, 38.38, 36.58, 29.55, 29.20, 28.34.LCMS 571.47 (M+H).

(3) Synthesis ofN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate

Dimethyl3-((2,2-dimethyl-4,20-dioxo-3,9,12,15-tetraoxa-5,19-diazahenicosan-21-yl)oxy)phthalate(1.589 g, 2.78 mmol, 1 eq) was dissolved in EtOH (14 mL, 0.2 M). Aqueous3M NaOH (2.8 mL, 8.34 mmol, 3 eq) was added and the mixture was heatedto 80° C. for 22 hours. The mixture was then cooled to room temperature,diluted with 50 mL DCM and 20 mL 0.5 M HCl. The layers were separatedand the organic layer was washed with 25 mL water. The aqueous layerswere combined and extracted three times with 50 mL chloroform. Thecombined organic layers were dried over sodium sulfate, filtered andcondensed to give 1.53 g of material that was carried forward withoutfurther purification. LCMS 553.44.

The resultant material (1.53 g) and 3-aminopiperidine-2,6-dionehydrochloride (0.480 g, 2.92 mmol, 1 eq) were dissolved in pyridine(11.7 mL, 0.25 M) and heated to 110° C. for 17 hours. The mixture wascooled to room temperature and concentrated under reduced pressure togive crude tert-butyl(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamateas a black sludge (3.1491 g) that was carried forward without furtherpurification. LCMS 635.47.

The crude tert-butyl(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamate(3.15 g) was dissolved in TFA (20 mL) and heated to 50° C. for 2.5hours. The mixture was cooled to room temperature, diluted with MeOH andconcentrated under reduced pressure. The material was purified bypreparative HPLC to giveN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate (1.2438 g, 1.9598 mmol, 71% over 3 steps) as a dark redoil.

¹H NMR (400 MHz, Methanol-d₄) δ 7.77 (dd, J=8.3, 7.5 Hz, 1H), 7.49 (d,J=7.3 Hz, 1H), 7.40 (d, J=8.5 Hz, 1H), 5.12 (dd, J=12.8, 5.5 Hz, 1H),4.75 (s, 2H), 3.68-3.51 (m, 12H), 3.40 (t, J=6.8 Hz, 2H), 3.10 (t, J=6.4Hz, 2H), 2.94-2.68 (m, 3H), 2.16 (dtd, J=12.6, 5.4, 2.5 Hz, 1H), 1.92(p, J=6.1 Hz, 2H), 1.86-1.77 (m, 2H). ¹³C NMR (100 MHz, cd₃od) δ 173.17,169.97, 168.48, 166.87, 166.30, 154.82, 136.89, 133.41, 120.29, 117.67,116.58, 69.96, 69.68, 69.60, 68.87, 68.12, 67.92, 49.19, 38.62, 36.14,30.80, 28.92, 26.63, 22.22. LCMS 536.41 (M+H).

(4) Synthesis of dFKBP-2

N-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate (12.5 mg, 0.0193 mmol, 1 eq) was added to SLF-succinate(12.08 mg, 0.0193 mmol, 1 eq) as a solution in 0.193 mL in DMF (0.1 M).DIPEA (10.1 microliters, 0.0580 mmol, 3 eq) and HATU (7.3 mg, 0.0193mmol, 1 eq) were added and the mixture was stirred for 19 hours. Themixture was then diluted with MeOH and purified by preparative HPLC togive dFKBP-2 (9.34 mg, 0.00818 mmol, 42%) as a yellow oil. ¹H NMR (400MHz, 50% MeOD/Chloroform-d) δ 7.76-7.70 (m, 1H), 7.58-7.45 (m, 3H), 7.26(t, J=8.2 Hz, 2H), 7.05-6.98 (m, 1H), 6.77 (d, J=7.9 Hz, 1H), 6.71-6.63(m, 2H), 5.73 (dd, J=8.1, 5.6 Hz, 1H), 5.23 (d, J=5.4 Hz, 1H), 5.03-4.95(m, 1H), 4.64 (s, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 3.62-3.52 (m, 8H),3.47 (t, J=6.1 Hz, 2H), 3.44-3.33 (m, 3H), 3.27-3.14 (m, 3H), 2.84-2.70(m, 3H), 2.64-2.47 (m, 6H), 2.34 (d, J=14.1 Hz, 1H), 2.24 (dd, J=14.3,9.3 Hz, 2H), 2.13-2.00 (m, 2H), 1.83 (p, J=6.3 Hz, 2H), 1.67 (dtd,J=38.4, 16.8, 14.8, 7.0 Hz, 7H), 1.51-1.26 (m, 3H), 1.22-1.05 (m, 6H),0.80 (dt, J=39.8, 7.5 Hz, 3H). ¹³C NMR (100 MHz, cdcl₃) δ 208.64,173.39, 173.01, 171.76, 170.11, 169.62, 168.24, 167.92, 167.36, 166.69,155.02, 149.23, 147.66, 140.94, 139.18, 137.57, 134.09, 133.91, 129.49,122.32, 120.75, 120.52, 119.93, 118.42, 117.75, 112.33, 111.98, 70.77,70.51, 70.40, 69.45, 69.04, 68.48, 56.20, 56.10, 51.88, 47.09, 44.78,38.40, 37.48, 36.91, 32.80, 32.71, 31.70, 31.59, 31.55, 29.53, 29.30,26.77, 25.22, 23.63, 23.33, 22.98, 21.43. LCMS 1141.71 (M+H).

Example 24: Synthesis of dFKBP-3

SLF-succinate was prepared according to step (1) of the synthesis ofdFKBP-1.

A 0.1 M solution ofN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate (0.233 mL, 0.0233 mmol, 1 eq) was added to2-(3-((R)-3-(3,4-dimethoxyphenyl)-1-(((S)-1-(3,3-dimethyl-2-oxopentanoyl)pyrrolidine-2-carbonyl)oxy)propyl)phenoxy)aceticacid (13.3 mg, 0.0233 mmol, 1 eq). DIPEA (12.2 microliters, 0.0700 mmol,3 eq) was added, followed by HATU (8.9 mg, 0.0233 mmol, 1 eq). Themixture was stirred for 23 hours, then diluted with MeOH and purified bypreparative HPLC to give a white solid (10.72 mg mg, 0.0112 mmol, 48%).

¹H NMR (400 MHz, Methanol-d₄) δ 7.79-7.74 (m, 1H), 7.52 (d, J=7.4 Hz,1H), 7.33 (d, J=8.4 Hz, 1H), 7.26 (t, J=8.1 Hz, 1H), 6.97-6.90 (m, 2H),6.89-6.84 (m, 1H), 6.79 (dd, J=8.2, 1.9 Hz, 1H), 6.73-6.64 (m, 2H),5.73-5.65 (m, 1H), 5.07-4.99 (m, 1H), 4.67 (s, 2H), 4.57-4.51 (m, 1H),4.48 (dd, J=5.7, 2.5 Hz, 2H), 3.82 (d, J=1.9 Hz, 3H), 3.80 (s, 3H),3.66-3.39 (m, 3H), 2.88-2.48 (m, 6H), 2.42-1.87 (m, 9H), 1.73-1.51 (m,6H), 1.19-0.92 (m, 6H), 0.75 (dt, J=56.7, 7.5 Hz, 3H). LCMS 954.52(M+H).

Example 25: Synthesis of dFKBP-4

SLF-succinate was prepared according to step (1) of the synthesis ofdFKBP-1.

A 0.1 M solution ofN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate (0.182 mL, 0.0182 mmol, 1 eq) was added to2-(3-((R)-3-(3,4-dimethoxyphenyl)-1-(((S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carbonyl)oxy)propyl)phenoxy)aceticacid (10.6 mg, 0.0182 mmol, 1 eq). DIPEA (9.5 microliters, 0.0545 mmol,3 eq) was added, followed by HATU (6.9 mg, 0.0182 mmol, 1 eq). Themixture was stirred for 26 hours, then diluted with MeOH and purified bypreparative HPLC to give a white solid (9.74 mg, 0.01006 mmol, 55%).

¹H NMR (400 MHz, Methanol-d₄) δ 7.75 (dd, J=8.3, 7.4 Hz, 1H), 7.53 (d,J=2.3 Hz, 1H), 7.33-7.25 (m, 2H), 7.00-6.84 (m, 3H), 6.79 (dd, J=8.1,2.5 Hz, 1H), 6.72-6.65 (m, 2H), 5.75-5.70 (m, 1H), 5.23 (d, J=4.9 Hz,1H), 5.05-4.96 (m, 1H), 4.66 (s, 2H), 4.46 (s, 2H), 3.82 (s, 3H), 3.81(s, 3H), 3.39-3.32 (m, 4H), 3.20-3.12 (m, 1H), 2.82-2.69 (m, 3H),2.62-2.49 (m, 2H), 2.37-2.00 (m, 5H), 1.78-1.30 (m, 11H), 1.24-1.08 (m,6H), 0.81 (dt, J=32.9, 7.5 Hz, 3H). LCMS 968.55 (M+H).

Example 26: Synthesis of dFKBP-5

SLF-succinate was prepared according to step (1) of the synthesis ofdFKBP-1.

A 0.1 M solution ofN-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate (0.205 mL, 0.0205 mmol, 1 eq) was added to2-(3-((R)-3-(3,4-dimethoxyphenyl)-1-(((S)-1-(2-phenylacetyl)piperidine-2-carbonyl)oxy)propyl)phenoxy)aceticacid (11.8 mg, 0.0205 mmol, 1 eq). DIPEA (10.7 microliters, 0.0615 mmol,3 eq) was added, followed by HATU (7.8 mg, 0.0205 mmol, 1 eq). Themixture was stirred for 29 hours, then diluted with MeOH and purified bypreparative HPLC to give a white solid (10.62 mg, 0.01106 mmol, 54%).

¹H NMR (400 MHz, Methanol-d₄) δ 7.77-7.72 (m, 1H), 7.52 (s, 1H),7.31-7.11 (m, 7H), 6.92-6.77 (m, 4H), 6.68-6.62 (m, 2H), 5.70-5.64 (m,1H), 5.38 (d, J=3.8 Hz, 1H), 4.99 (d, J=4.6 Hz, 1H), 4.65 (s, 2H),4.45-4.39 (m, 2H), 3.80 (dd, J=6.7, 2.4 Hz, 8H), 3.13-3.03 (m, 1H),2.83-2.68 (m, 3H), 2.63-2.45 (m, 3H), 2.34-1.93 (m, 6H), 1.71-1.52 (m,7H), 1.34-1.20 (m, 3H). LCMS 960.54 (M+H).

Example 27: Synthesis of diaminoethyl-acetyl-O-thalidomidetrifluoroacetate

(1) Synthesis of tert-Butyl (2-(2-chloroacetamido)ethyl)carbamate

tert-butyl (2-aminoethyl)carbamate (0.40 mL, 2.5 mmol, 1 eq) wasdissolved in THF (25 mL, 0.1 M) and DIPEA (0.44 mL, 2.5 mmol, 1 eq) at0° C. Chloroacetyl chloride (0.21 mL, 2.75 mmol, 1.1 eq) was added andthe mixture was allowed to warm to room temperature. After 22 hours, themixture was diluted with EtOAc and washed with saturated sodiumbicarbonate, water and brine. The organic layer was dried with sodiumsulfate, filtered and concentrated under reduced pressure to give awhite solid (0.66 g, quantitative yield) that carried forward to thenext step without further purification. ¹H NMR (400 MHz, Chloroform-d) δ7.16 (s, 1H), 4.83 (s, 1H), 4.04 (s, 2H), 3.42 (q, J=5.4 Hz, 2H), 3.32(q, J=5.6 Hz, 2H), 1.45 (s, 9H). LCMS 237.30 (M+H).

(2) Synthesis of dimethyl3-(2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-oxoethoxy)phthalate

tert-butyl (2-(2-chloroacetamido)ethyl)carbamate (0.66 g, 1 eq) wasdissolved in MeCN (17 mL, 0.15 M). Dimethyl 3-hydroxyphthalate (0.578 g,2.75 mmol, 1.1 eq) and cesium carbonate (2.24 g, 6.88 mmol, 2.75 eq)were then added. The flask was fitted with a reflux condenser and heatedto 80° C. for 32 hours. The mixture was then cooled to room temperature,diluted with EtOAc and washed three times with water. The organic layerwas dried over sodium sulfate, filtered and concentrated under reducedpressure. Purification by column chromatography (ISCO, 4 g silicacolumn, 0-15% MeOH/DCM over a 15 minute gradient) gave a yellow solid(0.394 g, 0.960 mmol, 38% over 2 steps). ¹H NMR (400 MHz, Chloroform-d)δ 7.65-7.56 (m, 1H), 7.50-7.41 (m, 1H), 7.27 (s, 1H), 7.11 (dd, J=8.4,4.1 Hz, 2H), 5.17 (s, 1H), 4.57 (d, J=6.3 Hz, 2H), 3.94 (s, 2H), 3.88(s, 2H), 3.40 (p, J=5.8 Hz, 4H), 3.32-3.19 (m, 4H), 1.39 (d, J=5.7 Hz,13H). ¹³C NMR (100 MHz, cdcl₃) δ 168.37, 168.23, 165.73, 156.13, 154.71,131.24, 130.09, 124.85, 123.49, 117.24, 79.42, 68.48, 53.22, 52.83,40.43, 39.54, 28.44. LCMS 411.45 (M+H).

(3) Synthesis of diaminoethyl-acetyl-O-thalidomide trifluoroacetate

Dimethyl3-(2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-oxoethoxy)phthalate(0.39 g, 0.970 mmol, 1 eq) was dissolved in EtOH (9.7 mL, 0.1 M).Aqueous 3M NaOH (0.97 mL, 2.91 mmol, 3 eq) was added and the mixture washeated to 80° C. for 3 hours. The mixture was cooled to roomtemperature, diluted with 50 mL DCM, 5 mL 1 M HCl and 20 mL water. Thelayers were separated and the organic layer was washed with 20 mL water.The combined aqueous layers were then extracted 3 times with 50 mLchloroform. The combined organic layers were dried over sodium sulfate,filtered and concentrated under reduced pressure to give a yellow solid(0.226 g) that was carried forward without further purification. LCMS383.36.

The resultant yellow solid (0.226 g) and 3-aminopiperidine-2,6-dionehydrochloride (0.102 g, 0.6197 mmol, 1 eq) were dissolved in pyridine(6.2 mL, 0.1 M) and heated to 110° C. for 16 hours. The mixture wascooled to room temperature and concentrated under reduced pressure togive tert-butyl(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethyl)carbamateas a poorly soluble black tar (0.663 g) which was carried forwardwithout purification (due to poor solubility). LCMS 475.42 (M+H).

The crude tert-butyl(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethyl)carbamatewas dissolved in TFA (10 mL) and heated to 50° C. for 3.5 hours, thenconcentrated under reduced pressure. Purification by preparative HPLCgave a red oil (176.7 mg, 0.362 mmol, 37% over 3 steps). ¹H NMR (400MHz, Methanol-d₄) δ 7.85-7.76 (m, 1H), 7.57-7.50 (m, 1H), 7.48-7.41 (m,1H), 5.13 (dd, J=12.6, 5.5 Hz, 1H), 4.81 (s, 2H), 3.62 (td, J=5.6, 1.8Hz, 2H), 3.14 (t, J=5.8 Hz, 2H), 2.97 (s, 1H), 2.80-2.66 (m, 2H), 2.15(dddd, J=10.1, 8.0, 5.8, 2.8 Hz, 1H). ¹³C NMR (100 MHz, cd₃od) δ 173.09,170.00, 169.99, 166.78, 166.62, 154.93, 136.88, 133.46, 120.71, 117.93,116.77, 68.29, 49.17, 39.37, 38.60, 30.73, 22.19. LCMS 375.30 (M+H forfree base).

Example 28: Synthesis of diaminobutyl-acetyl-O-thalidomidetrifluoroacetate

Diaminobutyl-acetyl-O-thalidomide trifluoroacetate was preparedaccording to the procedure in Fischer et al. Nature, 2014, 512, 49-53.

Example 29: Synthesis of diaminohexyl-acetyl-O-thalidomidetrifluoroacetate

(1) Synthesis of tert-butyl (6-(2-chloroacetamido)hexyl)carbamate

tert-butyl (6-aminohexyl)carbamate (0.224 mL, 1.0 mmol, 1 eq) wasdissolved in THE (10 mL, 0.1 M). DIPEA (0.17 mL, 1.0 mmol, 1 eq) wasadded and the mixture was cooled to 0° C. Chloroacetyl chloride (88microliters, 1.1 mmol, 1.1 eq) was added and the mixture was warmed toroom temperature and stirred for 18 hours. The mixture was then dilutedwith EtOAc and washed with saturated sodium bicarbonate, water andbrine. The organic layer was dried over sodium sulfate, filtered andconcentrated under reduced pressure to give a white solid (0.2691 g,0.919 mmol, 92%). ¹H NMR (400 MHz, Chloroform-d) δ 6.60 (s, 1H), 4.51(s, 1H), 4.05 (s, 2H), 3.30 (q, J=6.9 Hz, 2H), 3.11 (d, J=6.7 Hz, 2H),1.57-1.46 (m, 4H), 1.44 (s, 9H), 1.38-1.32 (m, 4H). LCMS 293.39 (M+H).

(2) Synthesis of dimethyl3-(2-((6-((tert-butoxycarbonyl)amino)hexyl)amino)-2-oxoethoxy)phthalate

tert-butyl (6-(2-chloroacetamido)hexyl)carbamate (0.2691 g, 0.919 mmol,1 eq) was dissolved in MeCN (9.2 mL, 0.1 M). Dimethyl 3-hydroxyphthalate(0.212 g, 1.01 mmol, 1.1 eq) and cesium carbonate (0.823 g, 2.53 mmol,2.75 eq) were added. The flask was fitted with a reflux condenser andheated to 80° C. for 14 hours. The mixture was cooled to roomtemperature and diluted with EtOAc, washed three times with water andback extracted once with EtOAc. The combined organic layers were driedover sodium sulfate, filtered and concentrated under reduced pressure.The crude material was purified by column chromatography (ISCO, 12 gsilica column, 0-15% MeOH/DCM 15 minute gradient) to give a yellow oil(0.304 g, 0.651 mmol, 71%). ¹H NMR (400 MHz, Chloroform-d) δ 7.66-7.58(m, 1H), 7.44 (td, J=8.2, 1.6 Hz, 1H), 7.15-7.08 (m, 1H), 6.96 (s, 1H),4.56 (s, 2H), 3.92 (t, J=1.6 Hz, 3H), 3.88 (t, J=1.6 Hz, 3H), 3.27 (q,J=6.9 Hz, 2H), 3.10-3.00 (m, 2H), 1.41 (s, 13H), 1.33-1.22 (m, 4H). ¹³CNMR (100 MHz, cdcl₃) δ 167.97, 167.37, 165.58, 155.95, 154.37, 130.97,129.74, 124.94, 123.26, 116.81, 78.96, 68.04, 52.89, 52.87, 52.69,52.67, 40.41, 38.96, 29.88, 29.13, 28.39, 26.33, 26.30. LCMS 467.49.

(3) Synthesis of diaminohexyl-acetyl-O-thalidomide trifluoroacetate

Dimethyl3-(2-((6-((tert-butoxycarbonyl)amino)hexyl)amino)-2-oxoethoxy)phthalate(0.304 g, 0.651 mmol, 1 eq) was dissolved in EtOH (6.5 mL, 0.1 M).Aqueous 3M NaOH (0.65 mL, 1.953 mmol, 3 eq) was added and the mixturewas heated to 80° C. for 18 hours. The mixture was cooled to roomtemperature and diluted with 50 mL DCM and 10 mL 0.5 M HCl. The layerswere separated and the organic layer was washed with 20 mL water. Thecombined aqueous layers were then extracted 3 times with chloroform. Thecombined organic layers were dried over sodium sulfate, filtered andconcentrated under reduced pressure to give a yellow foam (0.290 g) thatwas carried forward without further purification. LCMS 439.47.

The resultant yellow solid (0.290 g) and 3-aminopiperidine-2,6-dionehydrochloride (0.113 g, 0.69 mmol, 1 eq) were dissolved in pyridine (6.9mL, 0.1 M) and heated to 110° C. for 17 hours. The mixture was cooled toroom temperature and concentrated under reduced pressure to givetert-butyl(6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)hexyl)carbamateas a black solid (0.4216 g) which was carried forward withoutpurification (due to poor solubility). LCMS 531.41 (M+H).

The crude tert-butyl(6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)hexyl)carbamate(0.4216 g) was dissolved in TFA (10 mL) and heated to 50° C. for 2hours. The mixture was concentrated under reduced pressure, thenconcentrated under reduced pressure. Purification by preparative HPLCgave a brown solid (379.2 mg). ¹H NMR (400 MHz, Methanol-d₄) δ 7.79 (dd,J=8.4, 7.4 Hz, 1H), 7.52 (d, J=7.2 Hz, 1H), 7.42 (d, J=8.4 Hz, 1H), 5.13(dd, J=12.6, 5.5 Hz, 1H), 4.75 (s, 2H), 3.32 (t, J=7.6 Hz, 2H),2.96-2.89 (m, 2H), 2.89-2.65 (m, 3H), 2.16 (ddt, J=10.4, 5.4, 2.9 Hz,1H), 1.63 (dp, J=20.6, 7.1 Hz, 4H), 1.51-1.34 (m, 4H). ¹³C NMR (100 MHz,cd₃od) δ 174.57, 171.42, 169.90, 168.24, 167.79, 156.23, 138.23, 134.87,121.69, 119.22, 117.98, 69.36, 50.53, 40.64, 39.91, 32.14, 30.01, 28.44,27.23, 26.96, 23.63. LCMS 431.37 (M+H).

Example 30: Synthesis of diaminooctyl-acetyl-O-thalidomidetrifluoroacetate

(1) Synthesis of tert-Butyl (8-(2-chloroacetamido)octyl)carbamate

Octane-1,8-diamine (1.65 g, 11.45 mmol, 5 eq) was dissolved inchloroform (50 mL). A solution of di-tert-butyl dicarbonate (0.54 g,2.291 mmol, 1 eq) in chloroform (10 mL) was added slowly at roomtemperature and stirred for 16 hours before being concentrated underreduced pressure. The solid material was resuspended in a mixture ofDCM, MeOH, EtOAc and 0.5 N NH₃ (MeOH), filtered through celite andconcentrated under reduced pressure. Purification by columnchromatography (ISCO, 12 g NH2-silica column, 0-15% MeOH/DCM over a 15minute gradient) gave a mixture (1.75 g) of the desired product andstarting material which was carried forward without furtherpurification.

This mixture was dissolved in THE (72 mL) and DIPEA (1.25 mL, 7.16 mmol)and cooled to 0° C. Chloroacetyl chloride (0.63 mL, 7.88 mmol) was addedand the mixture was allowed to warm to room temperature. After 16 hours,the mixture was diluted with EtOAc and washed with saturated sodiumbicarbonate, water and brine. The resultant mixture was purified bycolumn chromatography (ISCO, dry load onto silica, 24 g column, 0-100%EtOAc/hexanes, over a 21 minute gradient) to give a white solid (0.56 g,1.745 mmol, 76% over 2 steps). ¹H NMR (400 MHz, Chloroform-d) δ 6.55 (s,1H), 4.48 (s, 1H), 4.05 (s, 2H), 3.30 (q, J=6.9 Hz, 2H), 3.10 (d, J=6.2Hz, 2H), 1.44 (s, 12H), 1.31 (s, 9H). ¹³C NMR (100 MHz, cdcl₃) δ 165.86,156.14, 77.36, 42.86, 40.73, 40.00, 30.18, 29.44, 29.26, 28.59, 26.86,26.82. LCMS 321.34 (M+H).

(2) Synthesis of dimethyl3-(2-((8-((tert-butoxycarbonyl)amino)octyl)amino)-2-oxoethoxy)phthalate

tert-butyl (8-(2-chloroacetamido)octyl)carbamate (0.468 g, 1.46 mmol, 1eq) was dissolved in MeCN (15 mL, 0.1 M). Dimethyl 3-hydroxyphthalate(0.337 g, 1.60 mmol, 1.1 eq) and cesium carbonate (1.308 g, 4.02 mmol,2.75 eq) were added. The flask was fitted with a reflux condenser andheated to 80° C. for 18 hours. The mixture was cooled to roomtemperature and diluted water and extracted once with chloroform andtwice with EtOAc. The combined organic layers were dried over sodiumsulfate, filtered and concentrated under reduced pressure.

The crude material was purified by column chromatography (ISCO, 24 gsilica column, 0-15% MeOH/DCM 20 minute gradient) to give a yellow oil(0.434 g, 0.878 mmol, 60%). 1H NMR (400 MHz, Chloroform-d) δ 7.57 (dd,J=7.9, 0.8 Hz, 1H), 7.40 (t, J=8.1 Hz, 1H), 7.07 (dd, J=8.4, 0.7 Hz,1H), 6.89 (t, J=5.3 Hz, 1H), 4.63 (s, 1H), 4.52 (s, 2H), 3.88 (s, 3H),3.83 (s, 3H), 3.22 (q, J=6.9 Hz, 2H), 3.01 (q, J=6.4 Hz, 2H), 1.36 (s,12H), 1.20 (s, 9H). ¹³C NMR (100 MHz, cdcl₃) δ 167.89, 167.29, 165.54,155.97, 154.38, 130.95, 129.69, 124.96, 123.23, 116.86, 78.82, 68.05,52.83, 52.82, 52.66, 52.64, 40.54, 39.06, 29.97, 29.19, 29.10, 29.06,28.40, 26.66, 26.61. LCMS 495.42 (M+H).

(3) Synthesis of diaminooctyl-acetyl-O-thalidomide trifluoroacetate

Dimethyl3-(2-((8-((tert-butoxycarbonyl)amino)octyl)amino)-2-oxoethoxy)phthalate(0.434 g, 0.878 mmol, 1 eq) was dissolved in EtOH (8.8 mL, 0.1 M)Aqueous 3M NaOH (0.88 mL, 2.63 mmol, 3 eq) was added and the mixture washeated to 80° C. for 24 hours. The mixture was cooled to roomtemperature and diluted with 50 mL DCM and 10 mL 0.5 M HCl. The layerswere separated and the organic layer was washed with 20 mL water. Thecombined aqueous layers were then extracted 3 times with chloroform. Thecombined organic layers were dried over sodium sulfate, filtered andconcentrated under reduced pressure to give a yellow solid (0.329 g)that was carried forward without further purification. LCMS 467.41.

The resultant yellow solid (0.329 g) and 3-aminopiperidine-2,6-dionehydrochloride (0.121 g, 0.734 mmol, 1 eq) were dissolved in pyridine(7.3 mL, 0.1 M) and heated to 110° C. for 20 hours. The mixture wascooled to room temperature and concentrated under reduced pressure togive tert-butyl(8-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)octyl) carbamate as a black tar (0.293 g) which was carried forwardwithout purification (due to poor solubility). LCMS 559.45 (M+H).

The crude tert-butyl(8-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)octyl)carbamate(0.293 g) was dissolved in TFA (10 mL) and heated to 50° C. for 4 hours.The mixture was concentrated under reduced pressure, then concentratedunder reduced pressure. Purification by preparative HPLC gave a brownresidue (114.69 mg, 23% over 3 steps). ¹H NMR (400 MHz, Methanol-d₄) δ7.84-7.78 (m, 1H), 7.54 (d, J=7.3 Hz, 1H), 7.43 (d, J=8.5 Hz, 1H), 5.13(dd, J=12.5, 5.5 Hz, 1H), 4.76 (s, 2H), 3.32 (d, J=4.1 Hz, 1H), 3.30 (d,J=3.3 Hz, 1H), 2.94-2.84 (m, 3H), 2.80-2.70 (m, 2H), 2.19-2.12 (m, 1H),1.67-1.55 (m, 4H), 1.40-1.34 (m, 8H). ¹³C NMR (100 MHz, cd₃od) δ 174.57,171.37, 169.85, 168.26, 167.78, 156.26, 138.22, 134.91, 121.70, 119.28,117.97, 69.37, 50.57, 40.76, 40.08, 32.17, 30.19, 30.05, 30.01, 28.52,27.68, 27.33, 23.63. LCMS 459.41 (M+H).

Example 31: Synthesis ofN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate

(1) Synthesis of tert-butyl(1-chloro-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamate

tert-butyl (3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate (1.0g, 3.12 mmol, 1 eq) was dissolved in THE (31 mL, 0.1 M). DIPEA (0.543mL, 3.12 mmol, 1 eq) was added and the solution was cooled to 0° C.Chloroacetyl chloride (0.273 mL, 3.43 mmool, 1.1 eq) was added and themixture was warmed slowly to room temperature. After 24 hours, themixture was diluted with EtOAc and washed with saturated sodiumbicarbonate, water then brine. The organic layer was dried over sodiumsulfate, filtered and condensed to give a yellow oil (1.416 g) that wascarried forward without further purification. ¹H NMR (400 MHz,Chloroform-d) δ 7.24 (s, 1H), 5.00 (s, 1H), 3.98-3.89 (m, 2H), 3.54(dddt, J=17.0, 11.2, 5.9, 2.2 Hz, 10H), 3.47-3.40 (m, 2H), 3.37-3.31 (m,2H), 3.17-3.07 (m, 2H), 1.79-1.70 (m, 2H), 1.67 (p, J=6.1 Hz, 2H), 1.35(s, 9H). ¹³C NMR (100 MHz, cdcl₃) δ 165.83, 155.97, 78.75, 70.49, 70.47,70.38, 70.30, 70.14, 69.48, 42.61, 38.62, 38.44, 29.62, 28.59, 28.40.LCMS 397.37 (M+H).

(2) Synthesis of dimethyl3-((2,2-dimethyl-4,20-dioxo-3,9,12,15-tetraoxa-5,19-diazahenicosan-21-yl)oxy)phthalate

tert-butyl(1-chloro-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamate(1.41 g, 3.12 mmol, 1 eq) was dissolved in MeCN (32 mL, 0.1 M). Dimethyl3-hydroxyphthalate (0.721 g, 3.43 mmol, 1.1 eq) and cesium carbonate(2.80 g, 8.58 mmol, 2.75 eq) were added. The flask was fitted with areflux condenser and heated to 80° C. for 19 hours. The mixture wascooled to room temperature and diluted water and extracted once withchloroform and twice with EtOAc. The combined organic layers were driedover sodium sulfate, filtered and concentrated under reduced pressure.The crude material was purified by column chromatography (ISCO, 24 gsilica column, 0-15% MeOH/DCM 22 minute gradient) to give a yellow oil(1.5892 g, 2.78 mmol, 89% over two steps). ¹H NMR (400 MHz,Chloroform-d) δ 7.52 (d, J=7.8 Hz, 1H), 7.35 (t, J=8.1 Hz, 1H), 7.04 (d,J=8.3 Hz, 1H), 7.00 (t, J=5.3 Hz, 1H), 5.06 (s, 1H), 4.46 (s, 2H), 3.83(s, 3H), 3.78 (s, 3H), 3.47 (ddd, J=14.9, 5.5, 2.8 Hz, 8H), 3.39 (dt,J=9.4, 6.0 Hz, 4H), 3.29 (q, J=6.5 Hz, 2H), 3.09 (d, J=6.0 Hz, 2H), 1.70(p, J=6.5 Hz, 2H), 1.63 (p, J=6.3 Hz, 2H), 1.31 (s, 9H). ¹³C NMR (100MHz, cdcl₃) δ 167.68, 167.36, 165.45, 155.93, 154.41, 130.87, 129.60,125.01, 123.20, 117.06, 78.60, 70.40, 70.17, 70.06, 69.39, 68.67, 68.25,52.77, 52.57, 38.38, 36.58, 29.55, 29.20, 28.34. LCMS 571.47 (M+H).

(3) Synthesis ofN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate

dimethyl3-((2,2-dimethyl-4,20-dioxo-3,9,12,15-tetraoxa-5,19-diazahenicosan-21-yl)oxy)phthalate(1.589 g, 2.78 mmol, 1 eq) was dissolved in EtOH (14 mL, 0.2 M). Aqueous3M NaOH (2.8 mL, 8.34 mmol, 3 eq) was added and the mixture was heatedto 80° C. for 22 hours. The mixture was then cooled to room temperature,diluted with 50 mL DCM and 20 mL 0.5 M HCl. The layers were separatedand the organic layer was washed with 25 mL water. The aqueous layerswere combined and extracted three times with 50 mL chloroform. Thecombined organic layers were dried over sodium sulfate, filtered andcondensed to give 1.53 g of material that was carried forward withoutfurther purification. LCMS 553.44.

The resultant material (1.53 g) and 3-aminopiperidine-2,6-dionehydrochloride (0.480 g, 2.92 mmol, 1 eq) were dissolved in pyridine(11.7 mL, 0.25 M) and heated to 110° C. for 17 hours. The mixture wascooled to room temperature and concentrated under reduced pressure togive crude tert-butyl(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamateas a black sludge (3.1491 g) that was carried forward without furtherpurification. LCMS 635.47.

The crude tert-butyl(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)carbamate(3.15 g) was dissolved in TFA (20 mL) and heated to 50° C. for 2.5hours. The mixture was cooled to room temperature, diluted with MeOH andconcentrated under reduced pressure. The material was purified bypreparative HPLC to giveN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamidetrifluoroacetate (1.2438 g, 1.9598 mmol, 71% over 3 steps) as a dark redoil. ¹H NMR (400 MHz, Methanol-d₄) δ 7.77 (dd, J=8.3, 7.5 Hz, 1H), 7.49(d, J=7.3 Hz, 1H), 7.40 (d, J=8.5 Hz, 1H), 5.12 (dd, J=12.8, 5.5 Hz,1H), 4.75 (s, 2H), 3.68-3.51 (m, 12H), 3.40 (t, J=6.8 Hz, 2H), 3.10 (t,J=6.4 Hz, 2H), 2.94-2.68 (m, 3H), 2.16 (dtd, J=12.6, 5.4, 2.5 Hz, 1H),1.92 (p, J=6.1 Hz, 2H), 1.86-1.77 (m, 2H). ¹³C NMR (100 MHz, cd₃od) δ173.17, 169.97, 168.48, 166.87, 166.30, 154.82, 136.89, 133.41, 120.29,117.67, 116.58, 69.96, 69.68, 69.60, 68.87, 68.12, 67.92, 49.19, 38.62,36.14, 30.80, 28.92, 26.63, 22.22. LCMS 536.41 (M+H).

Example 32: Synthesis ofN-(6-aminohexyl)-2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-carboxamide

(1) Synthesis of2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-carboxylic acid

1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxylic acid (0.192 g, 1 mmol, 1eq) and 3-aminopiperidine-2,6-dione hydrochloride (0.165 g, 1 mmol, 1eq) were dissolved in DMF (2.5 mL) and acetic acid (5 mL) and heated to80° C. for 24 hours. The mixture was then concentrated under reducedpressure and diluted with EtOH, from which a precipitate slowly formed.The precipitate was washed twice with EtOH to give a white solid (84.8mg, 0.28 mmol, 28%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.74 (s, 1H), 11.12(s, 1H), 8.39 (dd, J=7.8, 1.4 Hz, 1H), 8.26 (s, 1H), 8.04 (d, J=7.8 Hz,1H), 5.18 (dd, J=12.8, 5.4 Hz, 1H), 2.93-2.88 (m, 1H), 2.84 (d, J=4.7Hz, 0H), 2.66-2.50 (m, 2H), 2.12-1.99 (m, 1H). LCMS 303.19 (M+H).

(2) Synthesis of tert-butyl(6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-carboxamido)hexyl)carbamate

2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-carboxylic acid (22.7mg, 0.0751 mmol, 1 eq) and HATU (31.4 mg, 0.0826 mmol, 1.1 eq) weredissolved in DMF (0.75 mL). After 5 minutes, DIPA (39.2 microliters,0.225 mmol, 3 eq) was added. After an additional 5 minutes, tert-butyl(6-aminohexyl)carbamate (19.5 mg, 0.0901 mmol, 1.2 eq) was added as asolution in DMF (0.75 mL). The mixture was stirred for 20 hours, thendiluted with EtOAc. The organic layer was washed three times with brine,dried over sodium sulfate and concentrated under reduced pressure.Purification by column chromatography (ISCO, 4 g column, 0-10% MeOH/DCM,25 minute gradient) to give a yellow oil (17.18 mg, 0.03432 mmol, 46%).¹H NMR (400 MHz, Chloroform-d) δ 8.29 (d, J=6.2 Hz, 2H), 8.16 (s, 1H),7.94 (d, J=8.4 Hz, 1H), 6.91 (s, 1H), 5.00 (dd, J=12.4, 5.3 Hz, 1H),4.58 (s, 1H), 3.47 (q, J=6.7 Hz, 2H), 3.14 (q, J=8.5, 7.3 Hz, 2H),2.97-2.69 (m, 3H), 2.17 (ddd, J=10.4, 4.8, 2.6 Hz, 1H), 1.65 (p, J=6.9Hz, 2H), 1.53-1.32 (m, 15H). ¹³C NMR (100 MHz, cdcl₃) δ 174.69, 170.77,167.86, 166.67, 165.27, 156.49, 141.06, 133.95, 133.71, 132.13, 124.21,122.27, 77.36, 49.71, 39.75, 31.54, 30.27, 29.22, 28.57, 25.70, 25.37,22.73. LCMS 501.28 (M+H).

(3) Synthesis ofN-(6-aminohexyl)-2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-carboxamide

tert-butyl(6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-carboxamido)hexyl)carbamate(17.18 mg, 0.343 mmol, 1 eq) was dissolved in TFA (1 mL) and heated to50° C. for 2 hours. The mixture was concentrated under reduced pressureto give a yellow oil (13.29 mg) which was deemed sufficiently purewithout further purification. ¹H NMR (400 MHz, Methanol-d₄) δ 8.27 (dd,J=9.3, 1.3 Hz, 2H), 7.99 (d, J=7.6 Hz, 1H), 5.18 (dd, J=12.5, 5.4 Hz,1H), 3.48-3.40 (m, 2H), 2.96-2.84 (m, 3H), 2.76 (ddd, J=17.7, 8.1, 3.7Hz, 2H), 2.20-2.12 (m, 1H), 1.75-1.63 (m, 4H), 1.53-1.43 (m, 4H). LCMS401.31 (M+H).

Example 33: Synthesis of2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid

(1) Synthesis of2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione

4-hydroxyisobenzofuran-1,3-dione (0.773 g, 4.71 mmol, 1 eq) and3-aminopiperidine-2,6-dione hydrochloride (0.775 g, 4.71 mmol, 1 eq)were dissolved in pyridine (19 mL) and heated to 110° C. for 16 hours.The mixture was concentrated under reduced pressure and purified bycolumn chromatography (ISCO, 12 g silica column, 0-10% MeOH/DCM, 25minute gradient) to give an off white solid (1.14 g, 4.16 mmol, 88%). ¹HNMR (400 MHz, DMSO-d₆) δ 11.19 (s, 1H), 11.07 (s, 1H), 7.65 (dd, J=8.3,7.3 Hz, 1H), 7.31 (d, J=7.2 Hz, 1H), 7.24 (d, J=8.4 Hz, 1H), 5.07 (dd,J=12.8, 5.4 Hz, 1H), 2.88 (ddd, J=17.7, 14.2, 5.4 Hz, 1H), 2.63-2.50 (m,2H), 2.11-1.95 (m, 1H). LCMS 275.11 (M+H).

(2) Synthesis of tert-butyl2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetate

2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (218.8 mg,0.798 mmol, 1 eq) was dissolved in DMF (8 mL). Potassium carbonate(165.9 mg, 1.20 mmol, 1.5 eq) was added, followed by tert-butylbromoacetate (118 microliters, 0.798 mmol, 1 eq) and the mixture wasstirred at room temperature for 3 hours. The mixture was diluted withEtOAc and washed once with water and twice with brine. Purification bycolumn chromatography (ISCO, 12 g silica column, 0-100% EtOAc/hex, 17minute gradient) gave a white solid (0.26 g, 0.669 mmol, 84%). ¹H NMR(400 MHz, Chloroform-d) δ 8.74 (s, 1H), 7.61 (dd, J=8.4, 7.3 Hz, 1H),7.46-7.41 (m, 1H), 7.06 (d, J=8.3 Hz, 1H), 4.98-4.92 (m, 1H), 4.74 (s,2H), 2.83-2.69 (m, 3H), 2.12-2.04 (m, 1H), 1.43 (s, 9H). ¹³C NMR (100MHz, cdcl₃) δ 171.58, 168.37, 166.96, 166.87, 165.49, 155.45, 136.27,133.89, 119.78, 117.55, 116.83, 83.05, 66.52, 49.20, 31.37, 28.03,22.55. LCMS 411.23 (M+Na).

(3) Synthesis of2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid

tert-butyl2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetate(47.5 mg, 0.122 mmol, 1 eq) was dissolved in TFA (1.3 mL) at roomtemperature. After 3 hours, the mixture was diluted with DCM andconcentrated under reduced pressure to yield a white solid (42.27 mg),which was deemed sufficiently pure without further purification. ¹H NMR(400 MHz, Methanol-d₄) δ 7.76 (dd, J=8.5, 7.3 Hz, 1H), 7.50 (d, J=7.3Hz, 1H), 7.34 (d, J=8.5 Hz, 1H), 5.11 (dd, J=12.5, 5.5 Hz, 1H), 4.96 (s,2H), 2.87 (ddd, J=17.8, 14.2, 5.0 Hz, 1H), 2.80-2.65 (m, 2H), 2.18-2.09(m, 1H). LCMS 333.15 (M+H).

Example 34: dBET1 Treatment Downregulates BRD4 Levels

dBET1 showed potent binding to the first bromodomain of BRD4 (BRD4(1);IC₅₀=20 nM), while the epimeric dBET1(R) was comparatively inactive(IC₅₀=6.9 μM) (FIG. 1C). In comparison, the IC₅₀ JQ1 and JQ-R are 20 nMand 8.8 μM, respectively (FIG. 1C). Selectivity profiling confirmedpotent and BET-specific target engagement among 32 bromodomains studiedby phage display and displacement (FIG. 11A) (Table 2).

TABLE 2 Single Point Screening of dBET1 using BromoScan dBET1, 1 μMDiscoverRx Gene Symbol Percent Control ATAD2A 44 ATAD2B 62 BAZ2A 90BAZ2B 59 BRD1 50 BRD2(1) 0 BRD2(2) 0 BRD3(1) 0 BRD3(2) 0 BRD4(1) 0.25BRD4(2) 0 BRD7 96 BRD9 96 BRDT(1) 1.6 BRDT(2) 0 BRPF1 44 BRPF3 82 CECR265 CREBBP 32 EP300 30 FALZ 26 GCN5L2 96 PBRM1(2) 98 PBRM1(5) 100 PCAF 61SMARCA2 34 SMARCA4 47 TAF1(2) 56 TAF1L(2) 92 TRIM24(PHD,Bromo.) 100TRIM33(PHD,Bromo.) 100 WDR9(2) 36

A high-resolution crystal structure (1.0 Å) of dBET1 bound to BRD4(1)confirmed the mode of molecular recognition as comparable to JQ1 (FIGS.1D and 1G). Ordered density for the dBET1 ligand was found only to thefirst two carbon atoms of the butane spacer, suggesting conformationalflexibility of the conjugated phthalimide. Using the dBET1-BRD4(1)crystal structure and the recently reported structure of CRBN bound tothalidomide (E. S. Fischer et al., Nature 512, 49-53 (2014)), thefeasibility of ternary complex formation was modeled in silico. Anextended conformation of dBET1 was capable of bridging ordered BRD4(1)and CRBN in numerous sampled conformations without destructive stericinteractions (FIG. 1E). The modular nature of CRL complexes suggeststhat chemical recruitment of BRD4 may lead to CRBN-dependent degradation(E. S. Fischer et al., Cell 147, 1024-1039 (2011); M. D. Petroski, R. J.Deshaies, Nat. Rev. Mol. Cell Biol. 6, 9-2).

The chemical adaptor function of dBET1 was assessed by a homogeneousproximity assay for recombinant, human CRBN-DDB1 and BRD4 usingAlphaScreen technology. As shown in FIG. 11B, luminescence arising fromproximity of acceptor (CRBN-bound) and donor (BRD4-bound) beads wasincreased in the presence of low (10-100 nM) concentrations of dBET1. Athigher concentrations of dBET1 (e.g., 1 M), luminescence diminished,consistent with independent occupancy of CRBN and BRD4 binding sites byexcess dBET1 (the hook effect). Inhibition of chemical adaptor functionwas accomplished with competitive co-incubation with free JQ1 orthalidomide, each in a stereo-specific manner (FIG. 11C).

To functionally assess the effect of dBET1 on BRD4 in cells, a human AMLcell line (MV4-11) was treated for 18 hours with increasingconcentrations of dBET1 and assayed for endogenous BRD4 levels byimmunoblot. Pronounced loss of BRD4 (>85%) was observed withconcentrations of dBET1 as low as 100 nM (FIG. 1F). MV4-11 cells weretreated with DMSO or increasing concentrations of dBET1 for 24 hours,lysed in RIPA buffer. Immunoblotting was performed against the indicatedproteins. In addition, BRD4 and c-MYC levels were quantified relative tovinculin levels in cells treated with increasing concentrations of dBET1(FIG. 1H). Notably, JMJD6, a protein that physically interacts with BRD4was not affected (FIG. 1H). Moreover, HEXIM1 levels which usuallyincrease after BRD4 inhibitor treatment via a transcriptional controlmechanism were moderately decreased as well (FIG. 1H).

Molecular recognition of the BRD4 bromodomains by JQ1 isstereo-specific, and only the (+)-JQ1 enantiomer is active; the (−)-JQ1enantiomer (JQ1R) is inactive (FIGS. 1A-1C and FIG. 5 ). The epimericdBET1(R) compound lacks BRD4 binding (>300 fold weaker binding inhomogenous assays) and was inactive, demonstrating that BRD4 degradationrequires target protein engagement (FIG. 2A).

Example 35: Degradation of BRD2, BRD3, and BRD4 by dBET1

MV4-11 cells were treated with DMSO or 100 nM dBET1 for varioustimepoints between 1 and 24 hours. Cells were lysed in RIPA buffer.Immunoblotting was performed. While BRD3 and BRD4 showed comparablekinetics, BRD2 levels equilibrated faster at later timepoints. Theresults are shown in FIG. 2K.

To quantify dose-responsive effects on BRD4 protein stability, acell-count normalized, immunofluorescence-based high-content assay wasdeveloped in an adherent human cancer cell line (SUM149 breast cancercells (FIG. 2B). Potent downregulation of total BRD4 was observed fordBET1 (EC₅₀=430 nM) without apparent activity for dBET1(R). Theseobservations were confirmed by immunoblot in SUM149, which were used forbaseline normalization of the assay (FIG. 2C). Additional culturedadherent and non-adherent human cancer cell lines showed comparableresponse (SUM159, MOLM13) (FIGS. 2I and 2J).

The kinetics of BRD4 degradation were determined in a time courseexperiment using 100 nM dBET1 in MV4-11 cells. Marked depletion of BRD4was observed at 1 hour and complete degradation was observed at 2 hoursof treatment (FIG. 2D). Degradation of BRD4 by dBET1 was associated witha potent inhibitory effect on MV4-11 cell proliferation at 24 hours(measured by ATP content, IC₅₀=0.14 μM, compared to IC₅₀=1.1 μM (FIG.2E), consistent with the reported, pronounced inhibitory effect of RNAsilencing of BRD4 in this and other models of MLL-rearranged leukemia(J. Zuber et al., Nature 478, 524-528 (2011)). Moreover, dBET1 induced apotent apoptotic consequence in MV4-11 as measured by activated caspasecleavage (Caspase-GLO) (FIG. 2F), cleavage of poly(ADP-ribose)polymerase (PARP), and cleavage of Caspase-3 (FIG. 2N) and Annexin Vstaining (FIG. 2M). The apoptotic response to dBET1 was confirmed inadditional cultured human cell lines including DHL4 (B-cell lymphoma)(FIG. 2F). Kinetic studies of apoptotic response were then performed inMV4-11 cells cultured for either 4 or 8 hours followed by drug washout.Induction of apoptosis was assessed at 24 hours. While pulsed treatmentwith JQ1 did not yield a pronounced apoptotic response, significantlyincreased apoptosis was observed after only 4 h of dBET1 treatment thatwas enhanced at 8 h (FIGS. 2P and 2Q).

The rapid biochemical activity and robust apoptotic response of culturedcell lines to dBET1 established the feasibility of assessing effects onprimary human AML cells, where ex vivo proliferation is negligible inshort-term cultures. Exposure of primary leukemic patient blasts todBET1 elicited dose-proportionate depletion of BRD4 (immunoblot) (FIG.2G) and induction of apoptosis (Annexin V staining) (FIGS. 2H and 2L).Importantly, the effect of BRD4 degradation by dBET1 elicited asignificantly greater apoptotic response in primary AML cells and AMLcell lines than displacement of BRD4 by JQ1 (FIG. 2H). Together, thesedata demonstrated ligand-dependent target protein degradation andsupported that target degradation can elicit a more pronouncedbiological consequence than domain-specific target inhibition.

The therapeutic effect of BRD4 degradation was assessed in vivo byevaluating the tolerability and anti-tumor efficacy of repeat-dose dBET1in an established murine xenograft model of human MV4-11 leukemia cells.Tumor-bearing mice were treated with dBET1 administered byintraperitoneal injection (50 mg/kg daily) or vehicle control. After 14days of therapy a first tumor reached institutional limits for tumorsize, and the study was terminated for comparative assessment ofefficacy and modulation of BRD4 stability and function. Administrationof dBET1 attenuated tumor progression as determined by serial volumetricmeasurement (FIG. 12A), and decreased tumor weight assessed post-mortem(FIG. 12B). Acute pharmacodynamic degradation of BRD4 was observed fourhours after a first or second daily treatment with dBET1 (50 mg/kg IP)by immunoblot, accompanied by downregulation of MYC (FIG. 12C). Theresults were confirmed by quantitative immunohistochemistry for BRD4 andMYC following repeat-dose exposure to dBET1 for 14 days (FIG. 12D). Astatistically significant destabilization of BRD4, downregulation of MYCand inhibition of proliferation (Ki67 staining) was observed with dBET1compared to vehicle control in excised tumors (FIGS. 12D and 12E).Pharmacokinetic studies of dBET1 (50 mg/kg IP) corroborated adequatedrug exposure in vivo (Cmax=392 nM; FIG. 13B), above the EC₅₀ for BRD4knock-down observed in vitro (<100 nM). Notably, two weeks of dBET1 waswell tolerated by mice without a meaningful effect on weight, whiteblood count, hematocrit or platelet count (FIGS. 13C and 13D).

Example 36: Degradation is Specific for dBET1

To critically assess the mechanism of dBET1-induced BRD4 degradation,requirements on proteasome function, BRD4 binding, and CRBN binding,were examined using chemical genetic and gene editing approaches.Treatment with either JQ1 or thalidomide alone was insufficient toinduce BRD4 degradation in MV4-11 cells (FIGS. 3A and 3E). BRD4stability was rescued by pre-treatment with the irreversible proteasomeinhibitor carfilzomib (0.4 μM), indicating that proteasome function isrequired in dBET1-mediated BRD4 degradation (FIGS. 3B and 3F).Pre-treatment with excess JQ1 or thalidomide abolished dBET1-inducedBRD4 degradation, further confirming the requirement for both BRD4 andCRBN (FIGS. 3B and 3F). Cullin-based ubiquitin ligases requireneddylation of the cullin subunit for processive E3 ligase activity andtarget polyubiquitination (G. Lu, et al., Science 343, 305-309 (2014);R. I. Sufan, M. Ohh, Neoplasia 8, 956-963 (2006)). Pre-treatment withthe selective NAE1 inhibitor MLN4924(29) rescued BRD4 stability fromdBET1 exposure, further supporting dependence on active RING E3 ligaseactivity (FIG. 3C). Moreover, using a recently published human MM cellline (MM1.S-CRBN^(−/−)) that features an engineered knockout of CRBN byCRISPR/Cas9 technology (G. Lu, et al., Science 343, 305-309 (2014))confirmed the cellular requirement for CRBN (FIG. 3D). These data showedCRBN-dependent proteasomal degradation of BRD4 by dBET1.

Example 37: Highly Selective BET Bromodomain Degradation by ExpressionProteomics

An unbiased, proteome-wide approach was selected to assess the cellularconsequences of dBET1 treatment on protein stability. The acute impactof dBET1 treatment (250 nM) was compared to JQ1 (250 nM) and vehicle(DMSO 0.0025%) controls on protein stability in MV4-11 cells. A 2 hourincubation was selected to capture primary, immediate consequences ofsmall-molecule action and to mitigate expected, confounding effects onsuppressed transactivation of BRD4 target genes. Three biological samplereplicates were prepared for each treatment condition using isobarictagging that allowed the detection of 7429 proteins using a lowercut-off of at least two identified spectra per protein. Following BETbromodomain inhibition with JQ1, few proteomic changes are observed(FIG. 4A). Only MYC was significantly depleted by more than 2-fold after2 hours of JQ1 treatment, indicating the reported rapid and selectiveeffect of BET bromodomain inhibition on MYC expression in AML (FIGS. 4Aand 4C) (J. Zuber et al., Nature 478, 524-528 (2011)). JQ1 treatmentalso downregulated the oncoprotein PIM1 (FIGS. 4A and 4C).

Treatment with dBET1 elicited a comparable, modest effect on MYC andPIM1 expression. Only three additional proteins were identified assignificantly (p<0.001) and markedly (>5-fold) depleted in dBET1-treatedcells: BRD2, BRD3 and BRD4 (FIGS. 4B and 4C). Orthogonal detection ofBRD2, BRD3, BRD4, MYC and PIM1 was performed by immunoblot followingtreatment of MV4-11 leukemia cells with dBET1 or JQ1. BET family memberswere degraded only by dBET1, whereas MYC and PIM1 abundance wasdecreased by both dBET1 and JQ1, and to a lesser degree (FIG. 4D). Noeffect on Ikaros TF expression was observed in either treatmentcondition (FIG. 4F). Because MYC and PIM are often associated withmassive adjacent enhancer loci by epigenomic profiling (B. Chapuy etal., Cancer Cell 24, 777-790 (2013); J. Lovén et al., Cell 153, 320-334(2013)) suggestive of a transcriptional mechanism of downregulation,mRNA transcript abundance was measured for each depleted gene product(FIG. 4E). Treatment with either JQ1 or dBET1 downregulated MYC and PIMtranscription, suggestive of secondary transcriptional effects.Transcription of BRD4 and BRD3 were unaffected, consistent withpost-transcriptional effects. Transcription of BRD2 was affected by JQ1and dBET1, whereas protein stability of the BRD2 gene product was onlyinfluenced by dBET1, indicating transcriptional and post-transcriptionalconsequences. These data demonstrated a highly selective effect of dBET1on target protein stability, proteome-wide.

Example 38: Negative SAR JQ1-Rev (JQI-II-079)

MV4-11 cells were treated for 24 hours with either DMSO, dBET1 (100 nM)or the indicated concentrations of JQ1-REV (JQ-II-079), lysed with RIPAbuffer, and immunoblotted for BRD4 and Vinculin as loading control.While BRD4 levels were significantly decreased with dBET1 treatment,JQ1-Rev treatment did not yield any measureable effect. The results areshown in FIG. 5 .

Example 39: Decrease in BRD4 Protein Levels Proceed Measurable Decreasein BRD4 Transcript Levels

MV4-11 cells were treated with dBET1 or JQ1 for 2 and 4 hours at 100 nMor 1 uM each.

RNA was isolated using Qiagen RNAeasy kit and converted to cDNA usingVILO Superscript reverse transcriptase. BRD4 transcript levels wereassayed via qRT-PCR. The results are shown in FIG. 6 .

Example 40: dBET1 Mediated Degradation of BRD4 is Dependent on CRBNAvailability

Wild type MM1S proficient of CBRN expression (MM1S WT) as well asdeficient of CRBN expression (MM1S CRBN^(−/−)) were treated with dBET1for 8 hours. CRBN deficient isogenic cell line was reported elsewhere(Lu et al. Science 2014). Cells were lysed with RIPA buffer andimmunoblotted for BRD4 and tubulin as loading control. The results areshown in FIG. 7 .

Example 41: Effects of dBET2 on BRD4 Expression Levels

MV4-11 cells were treated with DMSO or increasing concentrations ofdBET2 for 8 hours, lysed in RIPA buffer and immunoblotting was performedagainst BRD4 and tubulin as loading control. The results are shown inFIG. 8 .

Example 42: Effects of dBET2 on PLK1 Expression Levels

(A) MV4-11 cells were treated with DMSO or increasing concentrations ofdBET2 for 8 hours, lysed in RIPA buffer and immunoblotting was performedagainst PLK1 and tubulin as loading control. (B) Quantification of (A).The intensity of the PLK1 bands was quantified to the respective tubulinloading control bands. The results are shown in FIG. 9 .

Example 43: dBET1 Mediated Degradation In Vivo

Short-term treatment studies were conducted with tumor-bearing miceusing the human MV4-11 leukemia murine xenograft model. Mice withestablished tumors were administered dBET1 (50 mg/kg), JQ1 (50 mg/kg) ora vehicle control, once daily for two days. Pharmacodynamic effects onBRD4 stability were determined 4 h after the second drug exposure, byimmunoblot. Treatment with dBET1 was associated with unambiguoussuppression of BRD4, compared to JQ1 and vehicle controls (FIG. 2O).Corroborating the pharmacologic advantage observed in cell lines andprimary patient cells, an increased apoptotic response following dBET1treatment in vivo was observed as measured by immunoblotting for PARPand caspase cleavage (FIG. 2O).

Example 44: dFKBP-1 and dFKBP-2 Mediated Degradation of FKBP12

Phthalimide-conjugated ligands to the cytosolic signaling protein,FKBP12, were synthesized. FKBP12 has been identified to play a role incardiac development, ryanodine receptor function, oncogenic signaling,and other biological phenotypes. At a known permissive site on theFKBP12-directed ligand AP1497, two chemical spacers were placed tocreate the conjugated phthalimides dFKBP-1 and dFKBP-2 (FIG. 10A). Bothpotently decreased FKBP12 abundance in MV4-11 cells (FIG. 10B), leadingto over 80% reduction of FKBP12 at 0.1 μM and 50% reduction at 0.01 μM,a 1000-fold improvement in potency as compared to conjugated PROTACligands which demonstrated activity at 25 μM. As with dBET1,destabilization of FKBP12 by dFKBP-1 was rescued by pre-treatment withcarfilzomib, MLN4924, free AP1497 or free thalidomide (FIG. 10C).CRBN-dependent degradation was established using previously publishedisogenic 293FT cell lines which are wild-type (293FT-WT) or deficient(293FT-CRBN-1-) for CRBN (G. Lu, et al., Science 343, 305-309 (2014)).Treatment of 293FT-WT cells with dFKBP-1 induced potent, dose-dependentdegradation of FKBP12, whereas 293FT-CRBN^(−/−) were unaffected (FIG.10D).

Example 45: Degradation of BRD Proteins by dBET

10,000 cells (293T WT or 293 CRBN−/−) were seeded per well using384-well plates. On the following day, dBET compounds were added atvarious concentrations. After being treated with the dBET compounds for4 hours, cells were fixed with formaledhyde, permeabilized using 0.1%triton, blocked with LiCor blocking buffer, and incubated with theprimary antibody (BRD4, 1:1000) overnight. On the following day, cellswere washed (5×TBST) and stained using Odysee Cell Stain (1:500). Asecondary antibody recoginizing the rabbit BRD4 antibody was addedsimultaneously (1:800). Images were quantified using LiCOR imager. BRD4levels in the cells after the dBET treatment were shown in FIGS. 14A-BB.

Various cells (BAF3_K-RAS, SEMK2, Monomac1, MM1S^(WT), MM1S^(CRBN−/−))were treated with increasing concentrations of dBET1 or dBET6 for −16hours. Cells were lysed and the lysates were immunoblotted to measurelevels of BRD4. The results are shown in FIGS. 15A-15D and 15F.

MV4-11 cells were treated with 50 nM dBET6 or 200 nM dBET18. For thefollowing 24 hours, levels of BRD4 or BRD4, BRD2 and BRD3 were detectedby immunoblotting at various time points. The results are shown in FIG.15E and FIG. 16B.

Example 46: Degradation of BRD and Other Proteins by dBET

293T WT or 293 CRBN−/− were treated with dBET2, dBET7, dBET8, or dBET10at 1 μM for 16 hours. The cells were then lysed and the lysates wereimmunoblotted to measure levels of BRD4 and PLK1. The results are shownin FIG. 16A.

Example 47: Viability of Cells Treated with dBET Compounds

Various cell lines (T-ALL (MOLT4, DND41, CUTLL1), LOUCY, MV4-11, andRS4-11) were plated in 384 well plates at 1000 cells/well. dBETcompounds were then added to the cells and incubated for 48 hours. ATPcontent was measured as a surrogate for cellular viability using ATPlite(Promega). The results were shown in FIGS. 17A-17E and FIG. 18A-18C.

Example 48: dGR Mediated Glucocorticoid Receptor Degradation

DND41 cells were grown in culture plates. dGR3 was added at variousconcentrations and incubated for 16 hours. The cells were then lysed,and the lysates were immunoblotted to measure GR levels. The results areshown in FIG. 19 .

Example 49: Experimental Procedures

Protein Purification and Crystal Structure

A construct of human BRD4 covering residues 44-168 in the pNIC28Bsa4vector (Addgene) was overexpressed in E. coli BL21 (DE3) in LB medium inthe presence of 50 mg/ml of kanamycin. Cells were grown at 37° C. to anOD of 0.8, induced with 500 μM isopropyl-1-thio-D-galactopyranoside,incubated overnight at 17° C., collected by centrifugation, and storedat −80° C. Cell pellets were sonicated in buffer A (50 mM hepes 7.5+300mM NaCl+10% glycerol+10 mM Imidazole+3 mM BME) and the resulting lysatewas centrifuged at 35,000 xg for 40 min. Ni-NTA beads (Qiagen) weremixed with lysate supernatant for 30 min and washed with buffer A. Beadswere transferred to an FPLC-compatible column and the bound protein waswashed with 15% buffer B (50 mM hepes 7.5+300 mM NaCl+10% glycerol+300mM Imidazole+3 mM BME) and eluted with 100% buffer B. TEV was added tothe eluted protein and incubated at 4° C. overnight. The sample was thenpassed through a desalting column (26/10 column) equilibrate with bufferA (without imidazole), and the eluted protein was subjected to a secondNi-NTA step to remove His-tag and TEV. The eluant was concentrated andpassed through a Superdex-200 10/300 column in 20 mM hepes7.5+150 mMNaCl+1 mM DTT. Fractions were pooled, concentrated to 14 mg/ml, andfrozen at −80° C.

Crystallization, Data Collection and Structure Determination

A 1.5-fold excess of 10 mM dBET1 (in DMSO) was mixed with 500 μM proteinand crystallized by sitting-drop vapor diffusion at 20° C. in thefollowing crystallization buffer: 15% PEG3350, 0.1 M Succinate. Crystalswere transferred briefly into crystallization buffer containing 25%glycerol prior to flash-freezing in liquid nitrogen. Diffraction datafrom complex crystals were collected at beamline 24ID-C of the NE-CAT atthe Advanced Photon Source (Argonne National Laboratory), and data-setswere integrated and scaled using XDS (Kabsch, W. Acta crystallographicaSection D, Biological crystallography 2010, 66, 133). Structures weresolved by molecular replacement using the program Phaser (Mccoy et al.,Journal of Applied Crystallography 2007, 40, 658) and the search modelPDB entry XXXX. The ligand was automatically positioned and refinedusing Buster (Smart et al. Acta crystallographica Section D, Biologicalcrystallography 2012, 68, 368). Iterative model building and refinementusing Phenix (Adams et al. Acta crystallographica Section D, Biologicalcrystallography 2010, 66, 213) and Coot (Emsley, P.; Cowtan, K. Actacrystallographica Section D, Biological crystallography 2004, 60, 2126)led to a model with statistics shown in Table 3.

TABLE 3 Data collection and refinement statistics. BRD4a/DB-2-190Wavelength (Ã) 0.9792 Resolution range (Ã) 34.31-0.99 (1.025-0.99) Spacegroup P 21 21 21 Unit cell 38.01 43.05 79.7 90 90 90 Total reflections440045 (17524)  Unique reflections 67392 (4589)  Multiplicity 6.5 (3.8)Completeness (%) 91.60 (63.09) Mean I/sigma(I) 21.20 (2.46)  WilsonB-factor 9.95 R-merge 0.04633 (0.6184)  R-meas 0.05011 CC1/2 0.999(0.742) CC*    1 (0.923) Reflections used for R-free R-work 0.1887(0.2620) R-free 0.1863 (0.2788) CC(work) CC(free) Number of non-hydrogenatoms 1333 macromolecules 1059 ligands 55 water 219 Protein residues 128RMS (bonds) 0.005 RMS (angles) 1.07 Ramachandran favored (%) 9.80E+01Ramachandran allowed (%) Ramachandran outliers (%) 0 Clashscore 4.63Average B-factor 14.5 macromolecules 12.4 ligands 22.8 solvent 22.8Statistics for the highest-resolution shell are shown in parentheses.BRD4 AlphaScreen

Assays were performed with minimal modifications from the manufacturer'sprotocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES,150 mMNaCl, 0.1% w/v BSA, 0.01% w/v Tween20, pH 7.5 and allowed toequilibrate to room temperature prior to addition to plates. Afteraddition of Alpha beads to master solutions all subsequent steps wereperformed under low light conditions. A 2× solution of components withfinal concentrations of BRD4 (see protein expression section) at 40 nM,Ni-coated Acceptor Bead at 10 g/mL, and 20 nM biotinylated-JQ1(Anders etal. Nature Biotechnology 2013, 32, 92) was added in 10 L to 384-wellplates (AlphaPlate-384, PerkinElmer, USA). Plates were spun down at150×g, 100 nL of compound in DMSO from stock plates were added by pintransfer using a Janus Workstation (PerkinElmer, USA). Thestreptavidin-coated donor beads (10 g/mL final) were added as withprevious the solution in a 2×, 10 L volume. Following this addition,plates were sealed with foil to prevent light exposure and evaporation.The plates were spun down again at 150×g. Plates were incubated at roomtemperature for 1 hour and then read on an Envision 2104 (PerkinElmer,USA) using the manufacturer's protocol.

CRBN-DDB1 Expression and Purification

Expression and purification of CRBN-DDB1 were performed as described inFischer, E. S. et al., Nature 512, 49 (2014), using Sf cells(Invitrogen). pFastBac vectors encoding human CRBN and DDB1 were usedfor expression of the proteins.

CRBN-DDB1/BRD4 Dimerization Assay

A bead-based AlphaScreen technology was used to detect CRBN-DDB1/BRD4dimerization by dBET1. In brief, GST-BRD4[49-170] (Sigma Aldrich) andCRBN-DDB1 were diluted to 125 nM and 250 nM, respectively, in assaybuffer (50 mM HEPES pH 7.4, 200 mM NaCl, 1 mM TCEP, and 0.1% BSA), and20 uL of protein mixture was added to each well of a 384-well AlphaPlate(PerkinElmer). Compounds were then added at 100 nL per well from DMSOstock plates using a CyBi®-Well vario pin tool. After 1 hr incubation atroom temperature, Nickel Chelate AlphaLISA® Acceptor and GlutathioneAlphaLISA® Donor beads (PerkinElmer) were diluted in assay buffer to a2× concentration (20 ug/ul) and added at 20 uL per well. Plates wereincubated for 1 hr at room temperature prior to luminescence detectionon an Envision 2104 plate reader (PerkinElmer).

For competition assays, GST-BRD4[49-170] and CRBN-DDB1 were diluted asabove in the presence of 111 nM dBET1. Compound addition and subsequentdetection was performed as described above.

Cell lines 293FT and 293FT^(CRBN−/−) were cultured in DMEM supplementedwith 10% FCS and 1% Penicillin/Streptomycin. MV4-11, MOLM13, MM1S andMM1S^(CRBN−/−) were cultured in RPMI supplemented with 10% FCS and 1%Penicillin/Streptomycin. SUM149 cells were cultured in HUMEC medium(cell application, 815-500) with DMEM F12 (coming cellgro, 10-090-CV)(1:1) and final 5% FCS with 1% Penicillin/Streptomycin.Culture of Primary Patient Material

Cells were freshly thawed and grown for 24 hours in StemSpan SFEM media(Stemcell) supplemented with (all in ng/ml final concentration): IL-3(20), IL-6 (20), FLT3L (100), SCF (100) and GSCF (20). After that, cellswere treated with dBET1 or JQ1 at the indicated concentrations withrenewed cytokines for 24 hours. Subsequently, cells were either used forimmunoblot analysis or for FACS analysis.

Analysis of Apoptotic Cells by Flow Cytometry

For each sample, cells were washed with 500 μL of PBS and spun down at400×g for 5 minutes and media aspirated off. Cells were then resuspendedin Annexin V binding buffer: 140 mMNaCl, 10 mM HEPES, 2.5 mM CaCl₂), pH7.4 and 500 μL of each sample transferred to 5 mL polystyrene FACS tubes(Falcon Cat. No. 352054). Cells were spun down at 400×g for 5 minutesand buffer aspirated off. To each sample, 400 μL of Annexin V bindingbuffer with 250 ng/mL FITC-Annexin V and 500 ng/mL propidium iodide wereadded for staining. Cells were then sorted on a BD LSRFortessa andanalyzed using FlowJo V10 software (Tree Star, Inc).

Analysis of Apoptotic Cells by Caspase Glo Assay

Caspaseglo assay (Promega) has been conducted following themanufacturer's recommendations. Cells were seeded at a density of 5000cells/well in a white 384 well plate (Thermo Scientific Nunc, #164610)in a total volume of 40 ul with respective compound or vehicle controltreatment. After a 24 h incubation, 30 ul of the Caspaseglo substratewere added per well. Plate was incubated in the dark for 90 minutes andread on Envision plate reader (Perkin Elmer).

BRD4-High Content Assay

SUM149 cells were plated at a density of 3×10⁵ cells/well using theinner 60 wells of a 96 well plate. 24 hours later, compounds were addedin at the respective concentrations. Assays were performed with minimalmodifications from the manufacturer's protocol (LICOR In-Cell WesternAssay Kit). In brief, cells were fixed in 3.7% formaldehyde in PBS for20 minutes on room temperature (200 ul per well) and subsequentlypermeabilized using 1×PBS with 0.1% Triton X-100 (5×200 ul per well).Then, cells were blocked using 100 ul of a 1:1 diluted Odyssey BlockingBuffer (LICOR) for 90 minutes on room temperature. Next, cells wereincubated with BRD4 antibody diluted 1:1000 in Odyssey Blocking Buffer(LICOR) (50 ul per well) overnight on 4° C. Next day, plate was washed 5times with TBST (200 ul per well). Then, plates are stained with a 1:800dilution of IRDye 800CW goat anti-Rabbit antibody (LICOR) andsimultaneously with CellTag 700 Stain (1:500, LICOR) for cellnormalization. Plates were incubated for 1 h in the dark on RT, washed 5times with TBST (200 ul per well) and imaged on the Odyssey CLx Imager(LI-COR).

qRT-PCR

RNA was isolated using RNeasy Plus Mini Kit (Qiagen) and 500 ng of totalRNA have been used per sample for reverse transcription usingSuperScript Reverse Transcriptase (Life Technologies). cDNA has beendiluted 1:9 and 2 ul have been used as template for qRT-PCR using SYBRSelect master mix. The following primers have been used:

SEQ ID NO. 1 GAPDH (F): CCACTCCTCCACCTTTGAC SEQ ID NO. 2GAPDH (R): ACCCTGTTGCTGTAGCCA SEQ ID NO. 3 BRD2 (F): GTGGTTCTCGGCGGTAAGSEQ ID NO. 4 BRD2 (R): GGTTGACACCCCGGATTAC SEQ ID NO. 5BRD3 (F): TTGGCAAACCTCATCTCAAA SEQ ID NO. 6BRD3 (R): GATGTCCGGCTGATGTTCTC SEQ ID NO. 7BRD4 (F): CTCCGCAGACATGCTAGTGA SEQ ID NO. 8BRD4 (R): GTAGGATGACTGGGCCTCTG SEQ ID NO. 9c-MYC (F): CACCGAGTCGTAGTCGAGGT SEQ ID NO. 10c-MYC (R): GCTGCTTAGACGCTGGATTT SEQ ID NO. 11PIM1 (F): TCATACAGCAGGATCCCCA SEQ ID NO. 12PIM1 (R): CCGTCTACACGGACTTCGATSample Preparation for Quantitative Mass Spectrometry Analysis

Sample were prepared as previously described (Weekes, M. P. et al., Cell157, 1460 (2014)) with the following modification. All solutions arereported as final concentrations. Lysis buffer (8 M Urea, 1% SDS, 50 mMTris pH 8.5, Protease and Phosphatase inhibitors from Roche) was addedto the cell pellets to achieve a cell lysate with a proteinconcentration between 2-8 mg/mL. A micro-BCA assay (Pierce) was used todetermine the final protein concentration in the cell lysate. Proteinswere reduced and alkylated as previously described. Proteins wereprecipitated using methanol/chloroform. In brief, four volumes ofmethanol was added to the cell lysate, followed by one volume ofchloroform, and finally three volumes of water. The mixture was vortexedand centrifuged to separate the chloroform phase from the aqueous phase.The precipitated protein was washed with one volume of ice coldmethanol. The washed precipitated protein was allowed to air dry.Precipitated protein was resuspended in 4 M Urea, 50 mM Tris pH 8.5.Proteins were first digested with LysC (1:50; enzyme:protein) for 12hours at 25° C. The LysC digestion is diluted down to 1 M Urea, 50 mMTris pH8.5 and then digested with trypsin (1:100; enzyme:protein) foranother 8 hours at 25° C. Peptides were desalted using a Cis solid phaseextraction cartridges. Dried peptides were resuspended in 200 mM EPPS,pH 8.0. Peptide quantification was performed using the micro-BCA assay(Pierce). The same amount of peptide from each condition was labeledwith tandem mass tag (TMT) reagent (1:4; peptide:TMT label) (Pierce).The 10-plex labeling reactions were performed for 2 hours at 25° C.Modification of tyrosine residue with TMT was reversed by the additionof 5% hydroxyl amine for 15 minutes at 25° C. The reaction was quenchedwith 0.5% TFA and samples were combined at a 1:1:1:1:1:1:1:1:1:1 ratio.Combined samples were desalted and offlinefractionated into 24 fractionsas previously described.

Liquid Chromatography-MS3 Spectrometry (LC-MS/MS)

12 of the 24 peptide fraction from the basic reverse phase step (everyother fraction) were analyzed with an LC-MS3 data collection strategy(McAlister, G. C. et al., Anal. Chem. 86, 7150 (2014)) on an OrbitrapFusion mass spectrometer (Thermo Fisher Scientific) equipped with aProxeon Easy nLC 1000 for online sample handling and peptideseparations. Approximately 5 g of peptide resuspended in 5% formicacid+5% acetonitrile was loaded onto a 100 m inner diameter fused-silicamicro capillary with a needle tip pulled to an internal diameter lessthan 5 μm. The column was packed in-house to a length of 35 cm with aCis reverse phase resin (GP118 resin 1.8 m, 120 Å, Sepax Technologies).The peptides were separated using a 120 min linear gradient from 3% to25% buffer B (100% ACN+0.125% formic acid) equilibrated with buffer A(3% ACN+0.125% formic acid) at a flow rate of 600 nL/min across thecolumn. The scan sequence for the Fusion Orbitrap began with an MS1spectrum (Orbitrap analysis, resolution 120,000, 400-1400 m/z scanrange, AGC target 2×105, maximum injection time 100 ms, dynamicexclusion of 75 seconds). “Top N” (the top 10 precursors) was selectedfor MS2 analysis, which consisted of CID (quadrupole isolation set at0.5 Da and ion trap analysis, AGC 4×103, NCE 35, maximum injection time150 ms). The top ten precursors from each MS2 scan were selected for MS3analysis (synchronous precursor selection), in which precursors werefragmented by HCD prior to Orbitrap analysis (NCE 55, max AGC 5×104,maximum injection time 150 ms, isolation window 2.5 Da, resolution60,000.

LC-MS3 Data Analysis

A suite of in-house software tools were used to for RAW file processingand controlling peptide and protein level false discovery rates,assembling proteins from peptides, and protein quantification frompeptides as previously described. MS/MS spectra were searched against aUniprot human database (February 2014) with both the forward and reversesequences. Database search criteria are as follows: tryptic with twomissed cleavages, a precursor mass tolerance of 50 ppm, fragment ionmass tolerance of 1.0 Da, static alkylation of cysteine (57.02146 Da),static TMT labeling of lysine residues and N-termini of peptides(229.162932 Da), and variable oxidation of methionine (15.99491 Da). TMTreporter ion intensities were measured using a 0.003 Da window aroundthe theoretical m/z for each reporter ion in the MS3 scan. Peptidespectral matches with poor quality MS3 spectra were excluded fromquantitation (<200 summed signal-to-noise across 10 channels and <0.5precursor isolation specificity).

MV4-11 Xenograft Experiment

1×10e7 MV4-11 cells were injected subcutaneously in a volume of 200 ulof PBS per mouse (NSG). Successful engraftment was monitored viabioluminescence and caliper measurement. 11 days post injection ofMV4-11 cells, tumors were palpable and mice were distributed in eitherthe control (vehicle) or the dBET1 treated groups. Mice were treatedonce daily with 50 mg/kg dBET1 or vehicle (captisol) via intraperitonealinjection. Tumor volume was recorded via caliper measurement. The studywas terminated 14 days post treatment start when the tumor size of avehicle treated mouse reached institutional limits.

Expression Proteomics

5×10⁶ cells have been treated with DMSO, 250 nM dBET1 or 250 nM JQ1 intriplicate for 2 hours, washed with 3 times with ice-cold PBS andsnap-frozen in liquid N2. Next, samples were mechanically homogenized inlysis buffer (8 M Urea, 1% SDS, 50 mM Tris, pH 8.5, protease andphosphatase inhibitors) and protein quantification was performed usingthe micro-BCA kit (Pierce). After protein quantification lysates wereimmediately reduced with DTT and alkylated with iodoacetimide. 600 μgprotein was precipitated by methanol/chloroform and digestion wasperformed using LysC and trypsin. 50 μg of each sample was labeled withTandem Mass Tag (TMT, Thermo Scientific) reagent and fractionated fortotal proteomic analysis.

Reverse-Phase fractionation was conducted under the followingconditions: Buffer A: 5% ACN, 50 mMAmBic pH 8.0, Buffer B: 90% ACN, 50mMAmBic pH 8.0 (Fraction size—37 seconds (˜300 μL)) Proteins werefractionated by bRP and collected into two sets of 12 fractions each.One complete set (12 fractions) from HPRP was analyzed on an OrbitrapFusion mass spectrometer. Peptides were separated using a gradient of 3to 25% acetonitrile in 0.125% formic acid over 200 minutes. Peptideswere detected (MS1) and quantified (MS3) in the Orbitrap, peptides weresequenced (MS2) in the ion trap.MS2 spectra were searched using theSEQUEST algorithm against a Uniprot composite database derived from thehuman proteome containing its reversed complement and knowncontaminants. All peptide spectral matches were filtered to a 1% falsediscovery rate (FDR) using the target-decoy strategy combined withlinear discriminant analysis. Proteins were quantified only frompeptides with a summed SN threshold of >=200 and MS2 isolationspecificity of 0.5.

Immunoblotting

Cells have been lysed using RIPA buffer supplemented with proteaseinhibitor cocktail (Roche) and 0.1% benzonase (Novagen) on ice for 15minutes. The lysates were spun at 16000×g for 15 minutes on 4° C. andprotein concentration was determined using BCA assay (Pierce). Thefollowing primary antibodies were used in this study: BRD2 (Bethyllabs), BRD3 (abcam), BRD4 (Bethyl labs), MYC, tubulin and vinculin (allSanta Cruz) as well as PIM1 (Cell Signaling Technology) and IKZF3 (NovusBiologicals). Blots were imaged using fluorescence-labeled secondaryantibodies (LI-COR) on the OdysseyCLxImager (LI-COR). Quantification ofband intensities has been performed using OdysseyCLx software (LI-COR).

Immunohistochemistry

BRD4 staining was performed using the A₃₀₁-985A antibody (Bethyl labs)following the recommended parameters at a concentration of 1:2000. MYCand Ki67 stainings were performed as described previously.Quantification of positively stained nuclei was conducted using theaperio software (Leica Biosystems).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the present application.

All patents, patent applications, and literature references cited hereinare hereby expressly incorporated by reference.

We claim:
 1. A compound, which is:

or a stereoisomer or pharmaceutically acceptable salt thereof, wherein:

is

Y is a bond, (CH₂)₁₋₆, (CH₂)₀₋₆—O, (CH₂)₀₋₆—C(O)NR₂′, (CH₂)₀₋₆—NR₂′C(O),(CH₂)₀₋₆—NH, or (CH₂)₀₋₆—NR₂; X is C(O) or C(R₃)₂; X₁-X₂ is C(R₃)═N orC(R₃)₂—C(R₃)₂; each R₁ is independently halogen, OH, C₁-C₆ alkyl, orC₁-C₆ alkoxy; R₂ is C₁-C₆ alkyl, C(O)—C₁-C₆ alkyl, or C(O)—C₃-C₆cycloalkyl; R₂′ is H or C₁-C₆ alkyl; each R₃ is independently H or C₁-C₃alkyl; each R₃′ is independently C₁-C₃ alkyl; each R₄ is independently Hor C₁-C₃ alkyl; or two R₄, together with the carbon atom to which theyare attached, form C(O); R₅ is H, deuterium, C₁-C₃ alkyl, F, or Cl; m is0, 1, 2, or 3; n is 0; the Linker is a group that covalently binds tothe Targeting Ligand and Y; and wherein the Targeting Ligand is:

wherein: T₇ is CH₂ or CH₂CH₂; Rg₁ is C(O)Rg₅ or (CH₂)₁₋₃Rg₆; nn10 is 0,1, 2, or 3; nn11 is 0, 1, 2, or 3; each Rg₂ is independently C₁-C₃alkyl, C₁-C₃ alkoxy, CN, or halogen; Rg₃ is C(O)NRg₄L, OL, NRg₄L, L,O—(CH₂)₁₋₃—C(O)NRg₄L, or NHC(O)—(CH₂)₁₋₃—C(O)NRg₄L; Rg₄ is H or C₁-C₃alkyl; Rg₅ is C₁₋₆ alkyl; Rg₆ is phenyl optionally substituted withC₁-C₃ alkyl, C₁-C₃ alkoxy, CN, or halogen; and L is the attachment pointto the linker.
 2. The compound of claim 1, wherein the Linker has theFormula:

wherein p1 is selected from 0, 1, 2, 3, 4, 5, and 6; p2 is selected from0, 1, 2, 3, 4, 5, and 6; p3 is selected from 0, 1, 2, 3, 4, and 5; eachW is independently absent, CH₂, O, S, NH or NR₅; Z is absent, CH₂, O, NHor N(C₁-C₃ alkyl); and Q is absent or —CH₂C(O)NH—; wherein the Linker iscovalently bonded to the Y with the

next to Q, and covalently bonded to the Targeting Ligand with the

next to Z.
 3. The compound of claim 2, wherein W is O or CH₂.
 4. Thecompound of claim 2, wherein Q is —CH₂C(O)NH—.
 5. The compound of claim1, wherein T₇ is CH₂.
 6. The compound of claim 5, wherein Rg₁ isC(O)Rg₅.
 7. The compound of claim 6, wherein Rg₂ is C₁-C₃ alkoxy.
 8. Thecompound of claim 1, wherein T₇ is CH₂CH₂.
 9. The compound of claim 8,wherein Rg₁ is C(O)Rg₅.
 10. The compound of claim 9, wherein Rg₂ isC₁-C₃ alkoxy.
 11. The compound of claim 1, wherein two R₄, together withthe carbon atom to which they are attached, form C(O).
 12. The compoundof claim 1, wherein

is


13. The compound of claim 12, wherein X is C(O).
 14. The compound ofclaim 12, wherein X is C(R₃)₂.
 15. The compound of claim 1, wherein R₃and R₅ are H.
 16. The compound of claim 1, wherein m is
 0. 17. Thecompound of claim 1, wherein nn10 is
 2. 18. The compound of claim 1,which is represented by:

or a stereoisomer or pharmaceutically acceptable salt thereof.
 19. Apharmaceutical composition comprising a therapeutically effective amountof the compound or stereoisomer or pharmaceutically acceptable saltthereof of claim 1, and a pharmaceutically acceptable carrier.
 20. Amethod for the treatment of Parkinson's disease, Alzheimer's disease ora cancer mediated by FKBP12, comprising administering a therapeuticallyeffective amount of the compound or stereoisomer or pharmaceuticallyacceptable salt thereof of claim 1, optionally in a pharmaceuticallyacceptable carrier, to a human in need thereof.
 21. The method of claim20, wherein the cancer is leukemia or breast cancer.